Neukinase, a downstream protein of neuregulin

ABSTRACT

The present invention relates to neukinase, a downstream protein kinase in the neuregulin signaling pathway. In certain aspects, the present invention provides isolated neukinase-encoding nucleic acids, neukinase polypeptides, oligonucleotides that hybridize to neukinase nucleic acids, and expression vectors containing neukinase-encoding sequences. The present invention further provides isolated host cells, antibodies, transgenic non-human animals, compositions, and kits relating to neukinase. In other aspects, the present invention further provides methods of identifying predisposition to cardiac dysfunction, methods of detecting the presence of neukinase, neukinase nucleic acid, methods of screening for agents which affect neukinase activity, and methods of modulating neukinase activity.

RELATED APPLICATIONS

This application is a divisional application of U.S patent applicationSer. No.: 11/894,542, filed Aug. 20, 2007, which claims the benefit ofU.S Provisional Application No 60/839,388, filed Aug. 21, 2006, and U.S.Provisional Application No 60/921,655, filed Apr. 2, 2007, the contentsof each of which are hereby incorporated by reference in theirentireties and for all purposes.

This application incorporates herein by reference a Sequence Listing asan ASCII text file entitled “11748-034-999_Sequence _Listing.txt”created on Jul. 18, 2017, and having a size of 35,245 bytes.

1. FIELD OF THE INVENTION

The present invention relates to neukinase, a downstream protein kinasein the neuregulin signaling pathway. In certain aspects, the presentinvention provides isolated neukinase-encoding nucleic acids, neukinasepolypeptides, oligonucleotides that hybridize to neukinase nucleicacids, and expression vectors containing neukinase-encoding sequences.The present invention further provides isolated host cells, antibodies,transgenic non-human animals, compositions, and kits relating toneukinase. In other aspects, the present invention further providesmethods of identifying predisposition to cardiac dysfunction, methods ofdetecting the presence of neukinase, neukinase nucleic acid, methods ofscreening for agents which affect neukinase activity, and methods ofmodulating neukinase activity.

2. BACKGROUND OF THE INVENTION

Heart failure affects approximately five million Americans, and morethan 550,000 new patients are diagnosed with the condition each year.Current drug therapy for heart failure is primarily directed toangiotensin-converting enzyme (ACE) inhibitors, which are vasodilatorsthat cause blood vessels to expand, lowering blood pressure and reducingthe heart's workload. While the percent reduction in mortality has beensignificant, the actual reduction in mortality with ACE inhibitors hasaveraged only 3%-4%, and there are several potential side effects.Additional limitations are associated with other options for preventingor treating heart failure. For example, heart transplantation is clearlymore expensive and invasive than drug treatment, and it is furtherlimited by the availability of donor hearts. Use of mechanical devices,such as biventricular pacemakers, are similarly invasive and expensive.Thus, there has been a need for new therapies given the deficiencies incurrent therapies.

One promising new therapy involves administration of neuregulin(hereinafter referred to as “NRG”) to a patient suffering from, or atrisk of developing, heart failure. NRGs comprise a family ofstructurally related growth and differentiation factors that includeNRG1, NRG2, NRG3 and NRG4 and isoforms thereof. For example, over 15distinct isoforms of NRG1 have been identified and divided into twolarge groups, known as α- and β-types, on the basis of differences inthe sequence of their essential epidermal growth factor (EGF)-likedomains. It has been shown that the EGF-like domains of NRG1, ranging insize from 50 to 64-amino acids, are sufficient to bind to and activatethese receptors. Previous studies have shown that neuregulin-1β (NRG-1β)can bind directly to ErbB3 and ErbB4 with high affinity.

Recent studies highlight the roles of NRG-1β, ErbB2 and ErbB4 in thecardiovascular development as well as in the maintenance of adult normalheart function. NRG-1β has been shown to enhance sarcomere organizationin adult cardiomyocytes. The short-term administration of a recombinantNRG-1β EGF-like domain significantly improves or protects againstdeterioration in myocardial performance in three distinct animal modelsof heart failure. More importantly. NRG-1β significantly prolongssurvival of animals experiencing heart failure. These effects makeNRG-1β promising as a broad spectrum therapeutic or lead compound forheart failure due to a variety of common diseases.

However, there is a need for additional methods of affecting neuregulinsignal transduction and/or activation of downstream neuregulin signalingtargets which can be used in a clinical setting for the prevention,treatment or delay of heart failure and/or cardiac hypertrophy.

3. SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of a novelprotein kinase, termed neukinase, which exhibits structural similarityto skeletal muscle myosin light chain kinase and acts as a downstreamcomponent in the neuregulin signaling pathway. A neukinase cDNA has beencloned and sequenced, and a neukinase amino acid sequence has beendetermined. The upstream regulatory protein, neuregulin, enhancesneukinase expression and/or phosphorylation, which in turn increasesphosphorylation of the downstream target myosin light chain. Asneukinase is highly expressed in heart tissue, the present inventionprovides a new mechanism underlying the prophylactic and therapeuticeffects of neuregulin on the heart, and identifies a new target fortreating cardiovascular disease.

Accordingly, in a first aspect, the present invention provides anisolated polypeptide comprising an amino acid sequence having at least70% identity to SEQ ID NO: 1, wherein the polypeptide is capable ofphosphorylating myosin light chain. In another aspect, the presentinvention provides an isolated polypeptide comprising an amino acidsequence having at least 70% identity to SEQ ID NO:2, wherein thepolypeptide is capable of phosphorylating myosin light chain. In certainembodiments, the isolated polypeptide is capable of phosphorylating themyosin light chain of cardiac myosin. In certain embodiments, theisolated polypeptide is capable of phosphorylating the myosin lightchain of cardiac myosin of a mammal, which includes but is not limitedto, a rat, mouse, or human. In one embodiment, the isolated polypeptidecomprises the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the isolated polypeptide comprises the amino acid sequenceof SEQ ID NO:2. In some embodiments, the isolated polypeptide comprisesthe amino acid sequence of SEQ ID NO:25.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence having at least70% identity to SEQ ID NO: 1. In certain embodiments, the isolatednucleic acid encodes a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1. In certain embodiments, the isolated nucleic acidcomprises a nucleic acid sequence having at least 70% identity to atleast about 500 contiguous nucleotides selected from SEQ ID NO:3 or thecomplement thereof. In certain embodiments, the isolated nucleic acidcomprises at least about 500 nucleotides selected from the nucleic acidsequence of SEQ ID NO:3, or the complement thereof. In a particularembodiment, the isolated nucleic acid comprises the nucleic acidsequence of SEQ ID NO:3, or the complement thereof.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence having at least70% identity to SEQ ID NO:2. In certain embodiments, the isolatednucleic acid encodes a polypeptide comprising the amino acid sequence ofSEQ ID NO:2. In some embodiments, the isolated nucleic acid encodes apolypeptide comprising an amino acid sequence having at least 70%identity to SEQ ID NO:25. In certain embodiments, the isolated nucleicacid encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:25. In certain embodiments, the isolated nucleic acid comprises anucleic acid sequence having at least 70% identity to at least about 500contiguous nucleotides selected from SEQ ID NO:4 or the complementthereof. In certain embodiments, the isolated nucleic acid comprises atleast about 500 nucleotides selected from the nucleic acid sequence ofSEQ ID NO:4, or the complement thereof. In a particular embodiment, theisolated nucleic acid comprises the nucleic acid sequence of SEQ IDNO:4, or the complement thereof.

In another aspect, the invention provides an isolated oligonucleotidecomprising at least about 10 consecutive nucleotides of SEQ ID NO:3 orits complementary strand. In another aspect, the invention provides anisolated oligonucleotide comprising at least about 10 consecutivenucleotides of SEQ ID NO:4 or its complementary strand. In certainembodiments, the isolated oligonucleotide comprises the nucleic acidsequence of SEQ ID NO:5 or SEQ ID NO:6.

In another aspect, the invention provides a vector comprising anisolated nucleic acid encoding a polypeptide, wherein the encodedpolypeptide is capable of phosphorylating myosin light chain. In certainembodiments, the vector comprises at least about 500 nucleotidesselected from the nucleic acid sequence of SEQ ID NO:3. In certainembodiments, the vector comprises the nucleic acid sequence of SEQ IDNO:3. In certain embodiments, the vector comprises at least about 500nucleotides selected from the nucleic acid sequence of SEQ ID NO:4. Incertain embodiments, the vector comprises the nucleic acid sequence ofSEQ ID NO:4. In a particular embodiment, the neukinase nucleic acidsequence in the vector is operably linked to a transcriptionalregulatory sequence. In certain embodiments, the vector is selected fromthe group comprising a plasmid, a cosmid, a virus, and a bacteriophage.In certain embodiments, the vector expresses a polypeptide comprisingSEQ ID NO: 1 in a cell transformed with said vector. In certainembodiments, the vector expresses a polypeptide comprising SEQ ID NO:2in a cell transformed with said vector. In certain embodiments, thevector expresses a polypeptide comprising SEQ ID NO:25 in a celltransformed with said vector.

In another aspect, the invention provides an isolated host cellcomprising a neukinase nucleic acid according to the present invention.In another aspect, the invention provides an isolated host cellcomprising a vector that expresses neukinase. In certain embodiments,the isolated host cell is a neonatal rat ventricular myocyte. In certainembodiments, the isolated host cell is an H9c2(2-1) cell.

In another aspect, the invention provides an antibody that specificallybinds to a polypeptide comprising an amino acid sequence of SEQ IDNO: 1. In another aspect, the invention provides an antibody thatspecifically binds to a polypeptide comprising an amino acid sequence ofSEQ ID NO:2. In some embodiments, the antibody specifically binds to aneukinase polypeptide comprising an amino acid sequence of SEQ ID NO:25.In certain embodiments, the antibody is a polyclonal, monoclonal, singlechain monoclonal, recombinant, chimeric, humanized, mammalian, or humanantibody.

In another aspect, the invention provides a transgenic non-human animal,which expresses a nucleic acid encoding neukinase polypeptide. Incertain embodiments, the transgenic non-human animal neukinasepolypeptide comprises the amino acid sequence of SEQ ID NO: 1. Incertain embodiments, the transgenic non-human animal neukinasepolypeptide comprises the amino acid sequence of SEQ ID NO:2. In certainembodiments, the transgenic non-human animal neukinase polypeptidecomprises the amino acid sequence of SEQ ID NO:25. In a particularembodiment, the transgenic non-human animal over- or under-expressesneukinase polypeptide. In one embodiment, the transgenic non-humananimal comprises a nucleic acid having at least 70% identity to SEQ IDNO:3, or the complement thereof. In another embodiment, the transgenicnon-human animal of comprises a nucleic acid having at least 70%identity to SEQ ID NO:4, or the complement thereof. In certainembodiments, the transgenic non-human animal is a mammal, including, butnot limited to, a mouse, rat, rabbit, hamster, or sheep.

In another aspect, the invention provides a transgenic non-human animalwhose germ cells comprise a homozygous null mutation in the endogenousnucleic acid sequence encoding neukinase, wherein the mutation iscreated by insertion of, e.g., a neomycin cassette, in reverseorientation to neukinase transcription and wherein said mutation hasbeen introduced into said animal by homologous recombination in anembryonic stem cell such that said animal does not express a functionalneukinase polypeptide. In certain embodiments, the transgenic non-humananimal is fertile and transmits said null mutation to its offspring. Inparticular embodiments, the transgenic non-human animal is a mammal,including, but not limited to, a mouse, rat, rabbit, hamster, or sheep.

In another aspect, the invention provides a method of screening foragents that affect neukinase activity, comprising: a) administering saidagent to a cell that expresses a neukinase polypeptide; and b) assessinga biological activity of the neukinase in the cell. In certainembodiments, the biological activity is selected from the groupconsisting of autoinhibition, phosphorylation of cardiac myosin, andexpression of neukinase.

In another aspect, the invention provides a method of screening foragents that affect neukinase activity, comprising: a) administering saidagent to a transgenic non-human animal according to the presentinvention; and b) assessing the animal for an alteration in cardiacfunction affected by said agent. In certain embodiments, the cardiacfunction is selected from the group consisting of interventricularseptum size, left ventricle end diastolic dimension, posterior wallthickness, left ventricle end systolic dimension, ejection fraction,fractional shortening, and cardiac cycle.

In another aspect, the invention provides a method of detecting thepresence of the neukinase nucleic acid in a sample, comprising: (a)contacting the sample with a nucleic acid that hybridizes to theneukinase nucleic acid; and (b) determining whether the nucleic acidbinds to a nucleic acid in the sample.

In another aspect, the invention provides a method for identifyingwhether a subject is genetically predisposed to cardiac dysfunction,comprising, detecting in a biological sample from the subject, aneukinase gene associated with cardiac dysfunction. In certainembodiments, the cardiac dysfunction is hypertrophic cardiomyopathy orheart failure.

In another aspect, the invention provides a composition comprising aneukinase polypeptide of the invention and a pharmaceutically acceptablecarrier. In another aspect, the invention provides a compositioncomprising a polypeptide having an amino acid sequence that comprisesSEQ ID NO: 1 and a pharmaceutically acceptable carrier. In anotheraspect, the invention provides a composition comprising a polypeptidehaving an amino acid sequence that comprises SEQ ID NO:2 and apharmaceutically acceptable carrier. In some embodiments, thecomposition comprises a polypeptide having an amino acid sequence thatcomprises SEQ ID NO:25 and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a composition comprising aneukinase-encoding nucleic acid of the invention and a pharmaceuticallyacceptable carrier. In another aspect, the invention provides acomposition comprising a polynucleotide encoding a polypeptide having anamino acid sequence that comprises SEQ ID NO: 1 and a pharmaceuticallyacceptable carrier. In certain embodiments, the polynucleotide comprisesa nucleotide sequence of SEQ ID NO:3. In another aspect, the inventionprovides a composition comprising a polynucleotide encoding apolypeptide having an amino acid sequence that comprises SEQ ID NO:2 anda pharmaceutically acceptable carrier. In some embodiments, thecomposition comprises a polynucleotide encoding a polypeptide having anamino acid sequence that comprises SEQ ID NO:25 and a pharmaceuticallyacceptable carrier. In certain embodiments, the polynucleotide comprisesa nucleotide sequence of SEQ ID NO:4.

In another aspect, the invention provides a kit comprising i) anisolated oligonucleotide comprising at least 10 consecutive nucleotidesof SEQ ID NO:3, or its complementary strand; and ii) a container. Incertain embodiments, the kit contains the oligonucleotide whichcomprises at least 15 consecutive nucleotides of SEQ ID NO:3 or itscomplementary strand.

In another aspect, the invention provides a kit comprising i) anisolated oligonucleotide comprising at least 10 consecutive nucleotidesof SEQ ID NO:4, or its complementary strand: and ii) a container. Incertain embodiments, the kit contains the oligonucleotide whichcomprises at least 15 consecutive nucleotides of SEQ ID NO:4, or itscomplementary strand.

In another aspect, the invention provides a method of modulatingneukinase activity, which comprises inhibiting the autoinhibitory domainof the neukinase polypeptide with a compound that inhibits such adomain. In certain embodiments, the compound is Ca²⁺/calmodulin.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Northern blot analysis of rat neukinase mRNA expression inrat heart, brain, spleen, lung, liver, skeletal muscle, kidney andtestis tissues. Hybridization with a β-actin specific probe serves as aloading control.

FIG. 2 shows Western blot analysis of human neukinase protein expressionin human gut, liver, heart, skeletal muscle, lung, kidney, uterus,spleen and thyroid tissues. The membrane was probed with ananti-neukinase rabbit polyclonal antibody. Probing with a anti-GAPDHantibody serves as a loading control.

FIG. 3 shows the levels of phosphorylated regulatory myosin light chain(RLC-P) in a cell-free neukinase activity assay. Recombinantly expressedneukinase and RLC were co-incubated in the presence or absence of Ca²⁺and calmodulin (CaM), −/+EGTA. RLC phosphorylation was assessed byWestern blot analysis using anti-RLC-P antibody as probe.

FIG. 4 shows an amino acid sequence alignment of rat neukinase (r.NK),human neukinase (h.NK) and human skeletal myosin light chain kinase(s.MLCK; accession no. NP_149109). Darkly shaded boxes representcompletely conserved residues, moderately shaded boxes representidentical residues, and lightly shaded boxes represent similar residues.The serine/threonine protein kinase catalytic domain of skeletal myosinlight chain kinase is underlined (residues 291-540).

5. DETAILED DESCRIPTION OF THE INVENTION

This disclosure provides, for the first time, an isolated cDNA moleculewhich, when transfected into cells can produce neukinase protein.Neukinase protein is believed to be linked to, inter alia, cardiacdysfunction, cardiac hypertrophy and certain forms of cardiomyopathysuch as hypertrophic cardiomyopathy and mid-cavitary ventricularhypertrophy. This disclosure provides the molecule, the nucleotidesequence of this cDNA and the amino acid sequence of neukinase proteinencoded by this cDNA.

Having herein provided the nucleotide sequence of the neukinase cDNA,correspondingly provided are the complementary DNA strands of the cDNAmolecule, and DNA molecules which hybridize under stringent conditionsto neukinase cDNA molecule, or its complementary strand. Suchhybridizing molecules include DNA molecules differing only by minorsequence changes, including nucleotide substitutions, deletions andadditions. Also comprehended by this invention are isolatedoligonucleotides comprising at least a portion of the cDNA molecule orits complementary strand. These oligonucleotides can be employed aseffective DNA hybridization probes or primers for use in the polymerasechain reaction. Such probes and primers are particularly useful in thescreening and diagnosis of persons genetically predisposed tohypertrophic cardiomyopathy and other forms of cardiac dysfunction, asthe result of neukinase gene mutations.

Recombinant DNA vector comprising the disclosed DNA molecules, andtransgenic host cells containing such recombinant vectors, are alsoprovided. Disclosed embodiments also include transgenic nonhuman animalswhich over- or under-express neukinase protein, or over- orunder-express fragments or variants of neukinase protein.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention hereinafter is divided into the subsectionsthat follow. All publications mentioned herein are incorporated byreference to disclose and describe the methods and/or materials inconnection with which the publications are cited.

5.1 Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, the singular forms “a,” “an,” and “the” mean “at leastone” or “one or more” unless the context clearly dictates otherwise.

As used herein, “neuregulin” or “NRG” used in the present inventionrefers to proteins or peptides that can bind and activate ErbB2. ErbB3,ErbB4 or combinations thereof, including but not limited to allneuregulin isoforms, neuregulin EGF domain alone, polypeptidescomprising neuregulin EGF-like domain, neuregulin mutants orderivatives, and any kind of neuregulin-like gene products that alsoactivate the above receptors as described in detail below. In preferredembodiments, neuregulin used in the present invention binds to andactivates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. Neuregulin alsoincludes NRG-1, NRG-2, NRG-3, and NRG-4 proteins, peptides, fragmentsand compounds that mimic the activities of neuregulin. Neuregulin usedin the present invention can activate the above ErbB receptors andmodulate their biological reactions, e.g., stimulate breast cancer celldifferentiation and milk protein secretion; induce the differentiationof neural crest cell into Schwann cell; stimulate acetylcholine receptorsynthesis in skeletal muscle cell; and/or improve cardiocytedifferentiation, survival and DNA synthesis. Neuregulin also includesthose variants with conservative amino acid substitutions that do notsubstantially alter their biological activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene. 4th ed., TheBenjamin/Cummings Pub. Co., p. 224 (1987)). Neuregulin proteinencompasses a neuregulin protein and peptide. Neuregulin nucleic acidencompasses neuregulin nucleic acid and neuregulin oligonucleotides.

As used herein, “epidermal growth factor-like domain” or “EGF-likedomain” refers to a polypeptide motif encoded by the neuregulin genethat binds to and activates ErbB2, ErbB3, ErbB4, or combinationsthereof, and bears a structural similarity to the EGF receptor-bindingdomain as disclosed in WO 00/64400, Holmes et al., Science,256:1205-1210 (1992): U.S. Pat. Nos. 5,530,109 and 5,716,930: Hijazi etal., Int. J. Oncol., 13:1061-1067 (1998): Chang et al., Nature,387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997);Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO 97/09425,the contents of which are all incorporated herein by reference. Incertain embodiments, EGF-like domain binds to and activates ErbB2/ErbB4or ErbB2/ErbB3 heterodimers. In certain embodiments, EGF-like domaincomprises the amino acid sequence of the receptor binding domain ofNRG-1. In some embodiments, EGF-like domain comprises the amino acidsequence corresponding to amino acid residues 177-226, 177-237, or177-240 of NRG-1. In certain embodiments, EGF-like domain comprises theamino acid sequence of the receptor binding domain of NRG-2. In certainembodiments, EGF-like domain comprises the amino acid sequence of thereceptor binding domain of NRG-3. In certain embodiments, EGF-likedomain comprises the amino acid sequence of the receptor binding domainof NRG-4. In certain embodiments, EGF-like domain comprises the aminoacid sequence of Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu CysPhe Met Val Lys Asp Leu Ser Asn Pro, as described in U.S. Pat. No.5,834,229.

As used herein, “neukinase” refers to proteins or peptides which have anamino acid sequence that is identical to SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:25, as well as proteins sharing sequence similarity, e.g.,70%, 75%, 80%, 85%, 90%, 95%, or greater percent identity, with theamino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:25.Further, these proteins have a biological activity in common with thepolypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2or SEQ ID NO:25, including, but not limited to, antigeniccross-reactivity, autoinhibition, phosphorylation activity, and thelike. It is also contemplated that a neukinase protein can have one ormore conservative or non-conservative amino acid substitutions, oradditions or deletions from the amino acid sequence of SEQ ID NO: 1, SEQID NO:2 or SEQ ID NO:25 so long as the protein having such sequencealteration shares a biological activity as described above with thepolypeptide of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:25. Neukinase alsoincludes proteins or peptides expressed from different mutations,different spliced forms and various sequence polymorphisms of theneukinase gene.

As used herein, “functional fragments and variants of neukinase” referto those fragments and variants that maintain one or more functions ofneukinase. It is recognized that the gene or cDNA encoding neukinase canbe considerably mutated without materially altering one or moreneukinase functions. First, the genetic code is well-known to bedegenerate, and thus different codons may encode the same amino acids.Second, even where an amino acid substitution is introduced, themutation can be conservative and have no material impact on theessential functions of neukinase. Third, part of the neukinasepolypeptide can be deleted without impairing or eliminating all of itsfunctions. Fourth, insertions or additions can be made in neukinase, forexample, adding epitope tags, without impairing or eliminating itsfunctions. Other modifications can be made without materially impairingone or more functions of neukinase, for example, in vivo or in vitrochemical and biochemical modifications which incorporate unusual aminoacids. Such modifications include, for example, acetylation,carboxylation, phosphorylation, glycosylation, ubiquination, labelingwith radionuclides, and various enzymatic modifications, as will bereadily appreciated by those skilled in the art. A variety of methodsfor labeling proteins and substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands.Functional fragments and variants can be of varying length. For example,some fragments have at least 10, 25, 50, 75, 100, or 200 or more aminoacid residues.

As used herein, “myosin light chain” refers to an about 18 kDa proteinwhich associates with the myosin heavy chain and participates in theregulation of myosin's force-generating ATPase activity. There are twomajor groupings of MLC: MLC1, sometimes referred to as the essentialmyosin light chain, abbreviated ELC; and MLC2, sometimes referred to asthe regulatory myosin light chain, abbreviated RLC. RLC is the primarybiological target of mvosin light chain kinase (MLCK)-mediatedphosphorylation. When phosphorylated by MLCK the phosphorylated form ofRLC is abbreviated of RLC-P. Isoforms of ELC and RLC existing inskeletal, smooth, and cardiac muscle have been described. As an example,the human cardiac RLC gene and cDNA are described by Macera et al.,Genomics 13: 829-31 (1992); (GenBank accession no. NM00432).

As used herein, a “functional fragment or variant of myosin light chain”refers to a polypeptide which is capable of being phosphorylated by aprotein having myosin light chain kinase biological activity. Itincludes any polypeptide six or more amino acid residues in length whichis capable of being phosphorylated by a protein having myosin lightchain kinase biological activity.

As used herein, “myosin light chain kinase biological activity” refersto the in vitro or in vivo enzymatic ability of a polypeptide or proteinto mediate covalent incorporation of a phosphate into a regulatorymyosin light chain. The term encompasses such enzymatic activityobserved with any isoform of MLCK (for example, smooth muscle, skeletalmuscle, and cardiac MLCK isoforms), as well as such enzymatic activityobserved with fragments and variants of MLCK isoforms (for example,naturally occurring mutants; mutations, insertions and deletionsintroduced through recombinant DNA techniques; and fragments generatedby proteolysis).

As used herein, “protein” is synonymous with “polypeptide” or “peptide”unless the context clearly dictates otherwise.

As used herein, a “neukinase gene” refers to a gene that encodesneukinase as defined herein. A mutation of neukinase gene includesnucleotide sequence changes, additions or deletions, including deletionof large portions or the entire neukinase gene, or duplications of allor substantially all of the gene. Alternatively, genetic expression ofneukinase can be deregulated such that neukinase is over or underexpressed. The term “neukinase gene” is understood to include thevarious sequence polymorphisms and allelic variations that exist withinthe population. This term relates primarily to an isolated codingsequence, but can also include some or all of the flanking regulatoryelements and/or intron sequences. The RNA transcribed from a mutantneukinase gene is mutant neukinase messenger RNA.

As used herein, “neukinase cDNA” refers to a cDNA molecule which, whentransfected or otherwise introduced into cells, expresses the neukinaseprotein. The neukinase cDNA can be derived, for instance, by reversetranscription from the mRNA encoded by the neukinase gene and lacksinternal non-coding segments and transcription regulatory sequencespresent in the neukinase gene. An exemplary human neukinase cDNA isshown as SEQ ID NO:4.

As used herein, “vector” refers to discrete elements that are used tointroduce heterologous DNA into cells for either expression orreplication thereof. Selection and use of such vehicles are well knownwithin the skill of the artisan. An expression vector includes vectorscapable of expressing DNA that are operatively linked with regulatorysequences, such as promoter regions, that are capable of effectingexpression of such DNA fragments. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, “transgenic animals” refers to non-human animals,preferably mammals, more preferably rodents such as rats or mice, inwhich one or more of the cells includes a transgene. Other transgenicanimals include primates, sheep, rabbits, hamsters, dogs, cows, goats,chickens, amphibians, etc. A “transgene” is exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops, and which remains in the genome of the mature animal.

As used herein, a “homologous recombinant animal” refers to a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which the endogenous neukinase gene has been altered by anexogenous DNA molecule that recombines homologously with endogenousneukinase in a (e.g., embryonic) cell prior to development of theanimal. Other homologous recombinant animals include rabbits, hamstersand sheep. Host cells with exogenous neukinase can be used to producenon-human transgenic animals, such as fertilized oocytes or embryonicstem cells into which neukinase-encoding sequences have been introduced.Such host cells can then be used to create non-human transgenic animalsor homologous recombinant animals.

As used herein, the term “biological sample” includes tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government, or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans.

As used herein, the term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which a therapeutic of the invention isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like.

As used herein, “ejection fraction” or “EF” means the portion of bloodthat is pumped out of a filled left ventricle (LV) as the result of aheartbeat. It may be defined by the following formula: (LV diastolicvolume−LV systolic volume)/LV diastolic volume.

As used herein, “fractional shortening” or “FS” means a ratio of thechange in the diameter of the left ventricle between the contracted andrelaxed states. It may be defined by the following formula: (LV enddiastolic diameter−LV end systolic diameter)/LV end diastolic diameter.

As used herein, “heart failure” means an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrhythmia, familial hypertrophic cardiomyopathy,ischemic heart disease, idiopathic dilated cardiomyopathy, myocarditisand the like. The heart failure can be caused by any number of factors,including, without limitation, ischemic, congenital, rheumatic, oridiopathic forms. Chronic cardiac hypertrophy is a significantlydiseased state which is a precursor to congestive heart failure andcardiac arrest.

As used herein, “myocardial infarction” refers to a blockade of acoronary artery or blood flow interruption leading to focal necrosis ofpart of the myocardium caused by severe and persistent ischemia.

As used herein, “ventricular muscle cell hypertrophy” is synonymous withcardiac hypertrophy and refers to a condition characterized by anincrease in the size of individual ventricular muscle cells, theincrease in cell size being sufficient to result in a clinical diagnosisof the patient or sufficient as to allow the cells to be determined aslarger (e.g., 2-fold or more larger than non-hypertrophic cells). It maybe accompanied by accumulation of contractile proteins within theindividual cardiac cells and activation of embryonic gene expression. Invitro and in vivo methods for determining the presence of ventricularmuscle cell hypertrophy are known to those skilled in the art. In vitroassays for ventricular muscle cell hypertrophy include those methodsdescribed herein, e.g., increased cell size and increased expression ofatrial natriuretic factor (ANF). Changes in cell size are used in ascoring system to determine the extent of hypertrophy. These changes canbe viewed with an inverted phase microscope, and the degree ofhypertrophy scored with an arbitrary scale of 7 to 0, with 7 being fullyhypertrophied cells, and 3 being non-stimulated cells. The 3 and 7states may be seen in Simpson et al., Circulation Res. 51: 787-801(1982), FIG. 2, A and B, respectively. The correlation betweenhypertrophy score and cell surface area (μm²) has been determined to belinear (correlation coefficient=0.99). In phenylephrine-inducedhypertrophy, non-exposed (normal) cells have a hypertrophy score of 3and a surface area/cell of 581 μm², and fully hypertrophied cells have ahypertrophy score of 7 and a surface area/cell of 1811 μm², orapproximately 200% of normal. Cells with a hypertrophy score of 4 have asurface area/cell of 771 μm², or approximately 30% greater size thannon-exposed cells; cells with a hypertrophy score of 5 have a surfacearea/cell of 1109 μm², or approximately 90% greater size thannon-exposed cells; and cells with a hypertrophy score of 6 have asurface area/cell of 1366 μm², or approximately 135% greater size thannon-exposed cells. The presence of ventricular muscle cell hypertrophypreferably includes cells exhibiting an increased size of about 15%(hypertrophy score 3.5) or more. Inducers of hypertrophy vary in theirability to induce a maximal hypertrophic response as scored by theabove-described assay. For example, the maximal increase in cell sizeinduced by endothelin is approximately a hypertrophy score of 5.

As used herein, “suppression” of ventricular muscle cell hypertrophymeans a reduction in one of the parameters indicating hypertrophyrelative to the hypertrophic condition, or a prevention of an increasein one of the parameters indicating hypertrophy relative to the normalcondition. For example, suppression of ventricular muscle cellhypertrophy can be measured as a reduction in cell size relative to thehypertrophic condition. Suppression of ventricular muscle cellhypertrophy means a decrease of cell size of 10/o or greater relative tothat observed in the hypertrophic condition. More preferably,suppression of hypertrophy means a decrease in cell size of 30% orgreater; most preferably, suppression of hypertrophy means a decrease ofcell size of 50% or more. Relative to the hypertrophy score assay whenphenylephrine is used as the inducing agent, these decreases wouldcorrelate with hypertrophy scores of about 6.5 or less, 5.0-5.5, and4.0-5.0, respectively. When a different agent is used as the inducingagent, suppression is measured relative to the maximum cell size (orhypertrophic score) measured in the presence of that inducer.

Prevention of ventricular muscle cell hypertrophy can be determined bypreventing an increase in cell size relative to normal cells, in thepresence of a concentration of inducer sufficient to fully inducehypertrophy. For example, prevention of hypertrophy means a cell sizeincrease less than 200% greater than non-induced cells in the presenceof maximally-stimulating concentration of inducer. More preferably,prevention of hypertrophy means a cell size increase less than 135%greater than non-induced cells; and most preferably, prevention ofhypertrophy means a cell size increase less than 90% greater thannon-induced cells. Relative to the hypertrophy score assay whenphenylephrine is used as the inducing agent, prevention of hypertrophyin the presence of a maximally-stimulating concentration ofphenylephrine means a hypertrophic score of about 6.0-6.5, 5.0-5.5, and4.0-4.5, respectively.

The in vivo determination of hypertrophy includes measurement ofcardiovascular parameters such as blood pressure, heart rate, systemicvascular resistance, contractility, force of heart beat, concentric ordilated hypertrophy, left ventricular systolic pressure, leftventricular mean pressure, left ventricular end-diastolic pressure,cardiac output, stroke index, histological parameters, and ventricularsize and wall thickness. Animal models available for determination ofdevelopment and suppression of ventricular muscle cell hypertrophy invivo include the pressure-overload mouse model, RV murine dysfunctionalmodel, transgenic mouse model, and post-myocardial infarction rat model.Medical methods for assessing the presence, development, and suppressionof ventricular muscle cell hypertrophy in human patients are known, andinclude, for example, measurements of diastolic and systolic parameters,estimates of ventricular mass, and pulmonary vein flows.

As used herein, an “effective amount” of an active agent for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. The amountmay cure the disease but, typically, is administered in order toameliorate the symptoms of the disease.

As used herein, “active agent” means any substance intended for thediagnosis, cure, mitigation, treatment, or prevention of disease inhumans and other animals, or to otherwise enhance physical and mentalwell being.

The terms “treatment.” “treating,” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect in a subject actively suffering from a condition. The effect maycompletely or partially treat a disease or symptom thereof and thus maybe therapeutic in terms of a partial or complete cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes inhibiting the disease, i.e., arresting itsdevelopment; or relieving the disease, i.e., causing regression of thedisease. In one example, treatment refers to treating patients with, orat risk for, development of heart disease and related conditions, e.g.,heart failure. More specifically, “treatment” is intended to meanproviding a therapeutically detectable and beneficial effect on apatient suffering from heart disease.

The terms “prevent,” “preventing,” and the like are used herein togenerally refer to preventing a disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas suffering from the disease. Thus, “prevent” can refer to prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) cardiac hypertrophy.

5.2 Polypeptides of the Invention

The present invention provides newly identified and isolatedpolypeptides referred to in the present application as rat neukinase andhuman neukinase, respectively. In some embodiments, the polypeptides arenative sequence rat and native sequence human neukinase polypeptides. Insome embodiments, the polypeptides comprise substantially the same aminoacid sequences as found in the native neukinase sequences. In certainembodiments, the invention provides amino acid sequences of functionalfragments and variants of neukinase that comprise an antigenicdeterminant (i.e., a portion of a polypeptide that can be recognized byan antibody) or which are otherwise functionally active, as well asnucleic acids encoding the foregoing. Neukinase functional activityencompasses one or more known functional activities associated with afull-length (wild-type) neukinase polypeptide, e.g., myosin light chainkinase biological activity; antigenicity (the ability to be bound by anantibody to a protein consisting of the amino acid sequence of SEQ IDNOS: 1 or 2); immunogenicity (the ability to induce the production of anantibody that binds SEQ ID NOS: 1 or 2), and so forth.

In some embodiments, the polypeptides comprise the amino acid sequenceshaving functionally inconsequential amino acid substitutions, and thushave amino acid sequences which differ from that of the native neukinasesequence. Substitutions can be introduced by mutation intoneukinase-encoding nucleic acid sequences that result in alterations inthe amino acid sequences of the encoded neukinase but do not alterneukinase function. For example, nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues can bemade in neukinase encoding sequences. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofneukinase without altering myosin light chain kinase biologicalactivity, whereas an “essential” amino acid residue is required for suchbiological activity. For example, amino acid residues that are conservedamong the neukinase polypeptides of the invention are predicted to beparticularly unsuitable for alteration. Amino acids for whichconservative substitutions can be made are well known in the art.

Useful conservative substitutions are shown in Table 1, “PreferredSubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the subject invention so long as the substitution does notmaterially alter the biological activity of the compound. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, indicated in Table 2 as exemplary are introducedand the products screened for neukinase polypeptide biological activity.

TABLE 1 Preferred Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys,Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) SerSer Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn,Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu(L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn ArgMet (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norleucine Leu

Non-conservative substitutions that effect: (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation; (2)the charge; (3) hydrophobicity; or (4) the bulk of the side chain of thetarget site, can modify neukinase polypeptide function or immunologicalidentity. Residues are divided into groups based on common side-chainproperties as denoted in Table 2. Non-conservative substitutions entailexchanging a member of one of these classes for another class.Substitutions may be introduced into conservative substitution sites ormore preferably into non-conserved sites.

TABLE 2 Amino acid classes Class Amino Acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (see Carter,Biochem. J. 237:1-7 (1986): Zoller and Smith, Methods Enzymol.154:329-50 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on cloned neukinase-encoding DNA to produceneukinase variant DNA (Ausubel et al., Current Protocols In MolecularBiology, John Wiley and Sons, New York (current edition); Sambrook etal., Molecular Cloning. A Laboratory Manual, 3d. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001).

In certain embodiments, neukinase used in the present invention includesneukinase mutants or derivatives having an amino acid substitution witha non-classical amino acid or chemical amino acid analog. Non-classicalamino acids include, but are not limited to, the D-isomers of the commonamino acids, α—amino isobutyric acid, 4—aminobutyric acid, Abu, 2—aminobutyric acid, γ-Abu, ε-Ahx, 6—amino hexanoic acid, Aib, 2—aminoisobutyric acid, 3—amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general.

In one embodiment, the present invention includes an isolatedpolypeptide comprising an amino acid sequence having at least 70%identity to SEQ ID NO: 1. In some embodiments, the polypeptide comprisesan amino acid sequence having at least 75%, 80%, 85%, 90%, or 95%identity to SEQ ID NO: 1. In a particular embodiment, the isolatedpolypeptide comprises the amino acid sequence of SEQ ID NO: 1.

The present invention also includes an isolated polypeptide comprisingan amino acid sequence having at least 70% identity to SEQ ID NO:2. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:2. In aparticular embodiment, the isolated polypeptide comprises the amino acidsequence of SEQ ID NO:2.

The present invention also includes an isolated polypeptide comprisingan amino acid sequence having at least 70% identity to SEQ ID NO:25. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:25. Ina particular embodiment, the isolated polypeptide comprises the aminoacid sequence of SEQ ID NO:25.

Percent identity in this context means the percentage of amino acidresidues in the candidate sequence that are identical (i.e., the aminoacid residues at a given position in the alignment are the same residue)or similar (i.e., the amino acid substitution at a given position in thealignment is a conservative substitution, as discussed above), to thecorresponding amino acid residue in the peptide after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence homology. In certain embodiments, a neukinase homologueis characterized by its percent sequence identity or percent sequencesimilarity with the naturally occurring neukinase sequence. Sequencehomology, including percentages of sequence identity and similarity, aredetermined using sequence alignment techniques well-known in the art,preferably computer algorithms designed for this purpose, using thedefault parameters of said computer algorithms or the software packagescontaining them.

Non-limiting examples of computer algorithms and software packagesincorporating such algorithms include the following. The BLAST family ofprograms exemplify a preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences (e.g., Karlin &Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified as inKarlin & Altschul, 1993, Proc. Natl. Acad Sci. USA 90:5873-5877).Altschul et al., 1990, J. Mol. Biol. 215:403-410, (describing NBLAST andXBLAST), Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402(describing Gapped BLAST, and PSI-Blast). Another preferred example isthe algorithm of Myers and Miller (1988 CABIOS 4:11-17) which isincorporated into the ALIGN program (version 2.0) and is available aspart of the GCG sequence alignment software package. Also preferred isthe FASTA program (Pearson W. R. and Lipman D. J., Proc. Nat. Acad. Sci.USA, 85:2444-2448, 1988), available as part of the Wisconsin SequenceAnalysis Package. Additional examples include BESTFIT, which uses the“local homology” algorithm of Smith and Waterman (Advances in AppliedMathematics, 2:482-489, 1981) to find best single region of similaritybetween two sequences, and which is preferable where the two sequencesbeing compared are dissimilar in length; and GAP, which aligns twosequences by finding a “maximum similarity” according to the algorithmof Neddleman and Wunsch (J. Mol. Biol. 48:443-354, 1970), and ispreferable where the two sequences are approximately the same length andan alignment is expected over the entire length.

Examples of homologues may be the ortholog proteins of other speciesincluding animals, plants, yeast, bacteria, and the like. Homologues mayalso be selected by, e.g., mutagenesis in a native protein. For example,homologues may be identified by site-specific mutagenesis in combinationwith assays for detecting protein-protein interactions. Additionalmethods, e.g., protein affinity chromatography, affinity blotting, invitro binding assays, and the like, will be apparent to skilled artisansapprised of the present invention.

For the purpose of comparing two different nucleic acid or polypeptidesequences, one sequence (test sequence) may be described to be aspecific “percent identical to” another sequence (reference sequence) inthe present disclosure. In this respect, when the length of the testsequence is less than 90% of the length of the reference sequence, thepercentage identity is determined by the algorithm of Myers and Miller,Bull. Math. Biol., 51:5-37 (1989) and Myers and Miller, Comput. Appl.Biosci., 4(1):11-17 (1988). Specifically, the identity is determined bythe ALIGN program. The default parameters can be used.

Where the length of the test sequence is at least 90% of the length ofthe reference sequence, the percentage identity is determined by thealgorithm of Karlin and Altschul, Proc. Natl. Acad Sci. USA, 90:5873-77(1993), which is incorporated into various BLAST programs. Specifically,the percentage identity is determined by the “BLAST 2 Sequences” tool.See Tatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999).For pairwise DNA-DNA comparison, the BLASTN 2.1.2 program is used withdefault parameters (Match: 1; Mismatch: −2; Open gap: 5 penalties;extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and wordsize: 11, with filter). For pairwise protein-protein sequencecomparison, the BLASTP 2.1.2 program is employed using defaultparameters (Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff:15; expect: 10.0; and wordsize: 3, with filter).

In certain embodiments, the isolated polypeptides of the presentinvention are capable of phosphorylating the myosin light chain ofcardiac mvosin. In certain embodiments, the isolated polypeptides of thepresent invention are capable of phosphorylating a functional fragmentor variant of myosin light chain of cardiac myosin. In certainembodiments, the isolated polypeptides are capable of phosphorylatingthe mvosin light chain, or functional fragments or variants of myosinlight chain, of cardiac myosin of a mammal. In certain embodiments, themammal is a rat, mouse or human. In some embodiments, the isolatedpolypeptides are capable of phosphorylating the myosin light chain, orfunctional fragments or variants of myosin light chain, of rat cardiacmyosin. In some embodiments, the isolated polypeptides are capable ofphosphorylating the myosin light chain, or functional fragments orvariants of myosin light chain, of mouse cardiac myosin. In particularembodiments, the isolated polypeptides are capable of phosphorylatingthe myosin light chain, or functional fragments or variants of myosinlight chain, of human cardiac myosin.

In some embodiments, the isolated polypeptides of the present inventionare capable of binding to, and can be activated by, Ca²⁺/calmodulin.Although not intending to be bound by any particular theory ofoperation, it is believed that activation of myosin light chain kinasesinvolves the binding of Ca²⁺/calmodulin to a calmodulin-binding sequencein a conserved regulatory segment of the polypeptide, which alsocontains an autoinhibitory sequence. Binding of Ca²⁺/calmodulin removesthe autoinhibitory sequence from the catalytic core of the polypeptide,wherein the active site is exposed for protein substrate binding andphosphorylation. Accordingly, in certain aspects, the present inventionprovides isolated polypeptides having the above properties.

5.3 Nucleic Acids of the Invention

In another aspect, the present invention provides newly identified andisolated nucleotide sequences encoding rat neukinase and human neukinaserespectively. In particular, nucleic acids encoding native sequence ratneukinase and native sequence human neukinase polypeptides have beenidentified and isolated.

The neukinase-encoding or related sequences provided by the instantinvention include those nucleotide sequences encoding substantially thesame amino acid sequences as found in native neukinase, as well as thoseencoded amino acid sequences having functionally inconsequential aminoacid substitutions, and thus have amino acid sequences which differ fromthat of the native sequence. Examples include the substitution of onebasic residue for another (i.e. Arg for Lys), the substitution of onehydrophobic residue for another (i.e. Leu for Ile), or the substitutionof one aromatic residue for another (i.e. Phe for Tyr, etc.).

The invention further relates to fragments of neukinase. Nucleic acidsencoding such fragments are thus also within the scope of the invention.The neukinase gene and neukinase-encoding nucleic acid sequences of theinvention include human and related genes (homologues) in other species.In some embodiments, the neukinase gene and neukinase-encoding nucleicacid sequences are from vertebrates, or more particularly, mammals. Insome embodiments, the neukinase gene and neukinase-encoding nucleic acidsequences are of rat origin. In a preferred embodiment of the invention,the neukinase gene and neukinase-encoding nucleic acid sequences are ofhuman origin.

In one aspect, the invention provides an isolated nucleic acid encodinga polypeptide comprising an amino acid sequence having at least 70%identity to SEQ ID NO: 1. In some embodiments, the nucleic acid encodesa polypeptide comprising an amino acid sequence having at least 75%,80%, 85%, 90%, or 95% identity to SEQ ID NO: 1. In a particularembodiment, the isolated nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the invention provides an isolated nucleic acidcomprising a nucleic acid sequence having at least 70% identity to atleast about 500 contiguous nucleotides selected from SEQ ID NO:3 or thecomplement thereof. In some embodiments, the nucleic acid comprises anucleic acid sequence having at least 70% identity to at least about500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or 2500contiguous nucleotides selected from SEQ ID NO:3. In some embodiments,the nucleic acid comprises a nucleic acid sequence having at least 75%identity to at least about 500 contiguous nucleotides selected from SEQID NO:3. In some embodiments, the nucleic acid comprises a nucleic acidsequence having at least 75% identity to at least about 500, 600, 700,800, 900, 1000, 1100, 1200, 1500, 2000, or 2500 contiguous nucleotidesselected from SEQ ID NO:3. In some embodiments, the nucleic acidcomprises a nucleic acid sequence having at least 80% identity to atleast about 500 contiguous nucleotides selected from SEQ ID NO:3. Insome embodiments, the nucleic acid comprises a nucleic acid sequencehaving at least 80% identity to at least about 500, 600, 700, 800, 900,1000, 1100, 1200, 1500, 2000, or 2500 contiguous nucleotides selectedfrom SEQ ID NO:3. In some embodiments, the nucleic acid comprises anucleic acid sequence having at least 85% identity to at least about 500contiguous nucleotides selected from SEQ ID NO:3. In some embodiments,the nucleic acid comprises a nucleic acid sequence having at least 85%identity to at least about 500, 600, 700, 800, 900, 1000, 1100, 1200,1500, 2000, or 2500 contiguous nucleotides selected from SEQ ID NO:3. Insome embodiments, the nucleic acid comprises a nucleic acid sequencehaving at least 90% identity to at least about 500 contiguousnucleotides selected from SEQ ID NO:3. In some embodiments, the nucleicacid comprises a nucleic acid sequence having at least 90% identity toat least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or2500 contiguous nucleotides selected from SEQ ID NO:3. In someembodiments, the nucleic acid comprises a nucleic acid sequence havingat least 95% identity to at least about 500 contiguous nucleotidesselected from SEQ ID NO:3. In some embodiments, the nucleic acidcomprises a nucleic acid sequence having at least 95% identity to atleast about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or2500 contiguous nucleotides selected from SEQ ID NO:3. In certainembodiments, the isolated nucleic acid comprises at least about 500nucleotides selected from the nucleic acid sequence of SEQ ID NO:3, orthe complement thereof. In certain embodiments, the isolated nucleicacid comprises at least about 500, 600, 700, 800, 900, 1000, 1100, 1200,1500, 2000, or 2500 nucleotides selected from the nucleic acid sequenceof SEQ ID NO:3, or the complement thereof. In a particular embodiment,the isolated nucleic acid comprises the nucleic acid sequence of SEQ IDNO:3, or the complement thereof.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence having at least70% identity to SEQ ID NO:2. In some embodiments, the nucleic acidencodes a polypeptide comprising an amino acid sequence having at least75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:2. In a particularembodiment, the isolated nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence having at least70% identity to SEQ ID NO:25. In some embodiments, the nucleic acidencodes a polypeptide comprising an amino acid sequence having at least75%, 80%. 85%, 90%, or 95% identity to SEQ ID NO:25. In a particularembodiment, the isolated nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:25.

In another aspect, the invention provides an isolated nucleic acidcomprising a nucleic acid sequence having at least 70% identity to atleast about 500 contiguous nucleotides selected from SEQ ID NO:4 or thecomplement thereof. In some embodiments, the nucleic acid comprises anucleic acid sequence having at least 70% identity to at least about500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or 2500contiguous nucleotides selected from SEQ ID NO:4. In some embodiments,the nucleic acid comprises a nucleic acid sequence having at least 75%identity to at least about 500 contiguous nucleotides selected from SEQID NO:4. In some embodiments, the nucleic acid comprises a nucleic acidsequence having at least 75% identity to at least about 500, 600, 700,800, 900, 1000, 1100, 1200, 1500, 2000, or 2500 contiguous nucleotidesselected from SEQ ID NO:4. In some embodiments, the nucleic acidcomprises a nucleic acid sequence having at least 80% identity to atleast about 500 contiguous nucleotides selected from SEQ ID NO:4. Insome embodiments, the nucleic acid comprises a nucleic acid sequencehaving at least 80% identity to at least about 500, 600, 700, 800, 900,1000, 1100, 1200, 1500, 2000, or 2500 contiguous nucleotides selectedfrom SEQ ID NO:4. In some embodiments, the nucleic acid comprises anucleic acid sequence having at least 85% identity to at least about 500contiguous nucleotides selected from SEQ ID NO:4. In some embodiments,the nucleic acid comprises a nucleic acid sequence having at least 85%identity to at least about 500, 600, 700, 800, 900, 1000, 1100, 1200,1500, 2000, or 2500 contiguous nucleotides selected from SEQ ID NO:4. Insome embodiments, the nucleic acid comprises a nucleic acid sequencehaving at least 90% identity to at least about 500 contiguousnucleotides selected from SEQ ID NO:4. In some embodiments, the nucleicacid comprises a nucleic acid sequence having at least 90% identity toat least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or2500 contiguous nucleotides selected from SEQ ID NO:4. In someembodiments, the nucleic acid comprises a nucleic acid sequence havingat least 95% identity to at least about 500 contiguous nucleotidesselected from SEQ ID NO:4. In some embodiments, the nucleic acidcomprises a nucleic acid sequence having at least 95% identity to atleast about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, or2500 contiguous nucleotides selected from SEQ ID NO:4. In certainembodiments, the isolated nucleic acid comprises at least about 500nucleotides selected from the nucleic acid sequence of SEQ ID NO:4, orthe complement thereof. In certain embodiments, the isolated nucleicacid comprises at least about 500, 600, 700, 800, 900, 1000, 1100, 1200,1500, 2000, or 2500 nucleotides selected from the nucleic acid sequenceof SEQ ID NO:4, or the complement thereof. In a particular embodiment,the isolated nucleic acid comprises the nucleic acid sequence of SEQ IDNO:4, or the complement thereof.

The present invention also includes nucleic acids that hybridize to orare complementary to the foregoing sequences. In specific aspects,nucleic acids are provided which comprise a sequence complementary to atleast 20, 30, 40, 50, 100, 200 nucleotides or the entire coding regionof neukinase, or the reverse complement (antisense) of any of thesesequences. In a specific embodiment, a nucleic acid which hybridizes toa neukinase nucleic acid sequence (e.g., having part or the whole ofsequence SEQ ID NO:3 or SEQ ID NO:4, or the complements thereof), underconditions of low stringency is provided. In some embodiments, saidnucleic acid corresponds to SEQ ID NO:7. In other embodiments, saidnucleic acid corresponds to SEQ ID NO:8, or a portion thereof.

By way of example and not limitation, procedures using such conditionsof low stringency are as follows (see also Shilo and Weinberg. 1981,Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792). Filters containing DNA canbe pretreated for 6 h at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations can becarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe can be used. Filterscan be incubated in hybridization mixture for 18-20 h at 40° C., andthen washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution can thenbe replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters may be blotted dry and exposed for autoradiography. Ifnecessary, filters may be washed for a third time at 65-68° C. andre-exposed to film. Other conditions of low stringency which may be usedare well known in the art (e.g., as employed for cross-specieshybridizations).

In another specific embodiment, a nucleic acid that hybridizes to anucleic acid encoding neukinase, or its reverse complement, underconditions of high stringency is provided. By way of example and notlimitation, procedures using such conditions of high stringency are asfollows. Prehybridization of filters containing DNA may be carried outfor 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. Filters may be hybridized for 48 hat 65° C. in prehybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing offilters may be done at 37° C. for 1 h in a solution containing 2×SSC,0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This can be followed by a washin 0.1×SSC at 50° C. for 45 minutes before autoradiography. Otherconditions of high stringency that may be used are well known in theart.

5.3.1 Cloning of the Neukinase Gene or cDNA

The present invention further provides methods and compositions relatingto the cloning of a gene or cDNA encoding neukinase. In one embodimentof the invention, expression cloning (a technique commonly known in theart), may be used to isolate a gene or cDNA encoding neukinase. Anexpression library may be constructed by any method known in the art. Inone embodiment, mRNA (e.g., human) is isolated, and cDNA is made andligated into an expression vector such that the cDNA is capable of beingexpressed by the host cell into which it is introduced. Variousscreening assays can then be used to select for the expressed neukinaseproduct. In one embodiment, anti-neukinase antibodies can be used forselection.

In another embodiment of the invention, polymerase chain reaction (PCR)may be used to amplify desired nucleic acid sequences of the presentinvention from a genomic or cDNA library. Isolated oligonucleotideprimers representing known neukinase-encoding sequences can be used asprimers in PCR. In certain embodiments, the isolated oligonucleotideprimer comprises at least 10 consecutive nucleotides of SEQ ID NO:3 orits complimentary strand. In certain embodiments, the isolatedoligonucleotide primer comprises at least 10 consecutive nucleotides ofSEQ ID NO:4 or its complimentary strand. In some embodiments, theisolated oligonucleotide primer comprises the nucleic acid sequence ofSEQ ID NO:5. In some embodiments, the isolated oligonucleotide primercomprises the nucleic acid sequence of SEQ ID NO:6. In a preferredaspect, the oligonucleotide primers represent at least part of theconserved segments of strong homology between neukinase-encoding genesof different species. The synthetic oligonucleotides may be utilized asprimers to amplify by PCR sequences from RNA or DNA, preferably a cDNAlibrary, of potential interest. Alternatively, one can synthesizedegenerate primers for use in the PCR reactions.

In the PCR reactions, the nucleic acid being amplified can include RNAor DNA, for example, mRNA, cDNA or genomic DNA from any eukaryoticspecies. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetusthermal cycler and Taq polymerase. It is also possible to vary thestringency of hybridization conditions used in priming the PCRreactions, to allow for greater or lesser degrees of nucleotide sequencesimilarity between a known neukinase nucleotide sequence and a nucleicacid homologue being isolated. For cross-species hybridization, lowstringency conditions are preferred. For same-species hybridization,moderately stringent conditions are preferred. After successfulamplification of a segment of a neukinase homologue, that segment may becloned, sequenced, and utilized as a probe to isolate a complete cDNA orgenomic clone. This, in turn, will permit the determination of thegene's complete nucleotide sequence, the analysis of its expression, andthe production of its protein product for functional analysis. In thisfashion, additional nucleotide sequences encoding neukinase or neukinasehomologues may be identified.

The above recited methods are not meant to limit the following generaldescription of methods by which clones of genes encoding neukinase orhomologues thereof may be obtained.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the neukinase gene, neukinase cDNA or ahomologue thereof. The nucleic acid sequences encoding neukinase can beisolated from vertebrate, mammalian, human, porcine, bovine, feline,avian, equine, canine, as well as additional primate sources. The DNAmay be obtained by standard procedures known in the art from cloned DNA(e.g., a DNA “library”), by chemical synthesis, by cDNA cloning, or bythe cloning of genomic DNA, or fragments thereof, purified from thedesired cell, or by PCR amplification and cloning. See, for example,Sambrook et al., Molecular Cloning. A Laboratory Manual, 3d. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Glover,D. M. (ed.), DNA Cloning: A Practical Approach, 2d. ed., MRL Press,Ltd., Oxford, U.K. (1995). Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. Whatever the source,the gene may be cloned into a suitable vector for propagation of thegene.

In the cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNase in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, if a neukinase gene (of any species) or its specificRNA is available and can be purified and labeled, the generated DNAfragments may be screened by nucleic acid hybridization to the labeledprobe (Benton and Davis, Science 196:180 (1977); Grunstein and Hogness.Proc. Natl. Acad. Sci. U.S.A. 72:3961 (1975). Those DNA fragments withsubstantial homology to the probe will hybridize. It is also possible toidentify the appropriate fragment by restriction enzyme digestion(s) andcomparison of fragment sizes with those expected according to a knownrestriction map if such is available. Further selection can be carriedout on the basis of the properties of the gene.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones that hybrid-select theproper mRNAs, can be selected that produce a protein having e.g.,similar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, substrate binding activity, orantigenic properties as known for a specific neukinase. If an antibodyto a particular neukinase is available, that neukinase may be identifiedby binding of labeled antibody to the clone(s) putatively producing theneukinase in an ELISA (enzyme-linked immunosorbent assay)-typeprocedure.

A neukinase or homologue thereof can also be identified by mRNAselection by nucleic acid hybridization followed by in vitrotranslation. In this procedure, fragments are used to isolatecomplementary mRNAs by hybridization. Such DNA fragments may representavailable, purified DNA of another species containing a gene encodingneukinase. Immunoprecipitation analysis or functional assays of the invitro translation products of the isolated mRNAs identifies the mRNAand, therefore, the complementary DNA fragments that contain the desiredsequences. In addition, specific mRNAs may be selected by adsorption ofpolysomes isolated from cells to immobilized antibodies specificallydirected against a specific neukinase. A radiolabelledneukinase-encoding cDNA can be synthesized using the selected mRNA (fromthe adsorbed polysomes) as a template. The radiolabelled mRNA or cDNAmay then be used as a probe to identify the neukinase-encoding DNAfragments from among other genomic DNA fragments.

Alternatives to isolating the neukinase genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes neukinase. Forexample RNA for the cloning of neukinase cDNA can be isolated from cellsthat express a neukinase gene. Other methods are possible and within thescope of the invention.

The identified and isolated neukinase or neukinase analog-encoding genecan then be inserted into an appropriate cloning vector. A large numberof vector-host systems known in the art may be used. Possible cloningvectors include, but are not limited to, plasmids or modified viruses,but the vector system must be compatible with the host cell used. Suchvectors include, but are not limited to bacteriophages such as lambdaderivatives, or plasmids such as pBR322, pUC plasmid derivatives, or thepBluescript vector. (Stratagene). The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini. Theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector andneukinase-encoding gene or nucleic acid sequence may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shotgun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

To generate multiple copies of the isolated neukinase-encoding gene,cDNA, or synthesized DNA sequence, host cells, for example competentstrains of E. Coli, may be transformed with recombinant DNA moleculesincorporating said sequences according to any technique known in theart. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

5.3.2 Expression Vectors

In still another aspect, the invention provides expression vectors forexpressing isolated neukinase-encoding cDNA sequences. Generally,expression vectors are recombinant polynucleotide molecules comprisingexpression control sequences operatively linked to a nucleotide sequenceencoding a polypeptide. Expression vectors can readily be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, selectable markers, etc. to result instable transcription and translation of mRNA. Techniques forconstruction of expression vectors and expression of genes in cellscomprising the expression vectors are well known in the art. See, e.g.,Sambrook et al., 2001, Molecular Cloning—A Laboratory Manual, 3^(rd)edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. andAusubel et al., eds., Current Edition, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

Useful promoters for use in expression vectors include, but are notlimited to, a metallothionein promoter, a constitutive adenovirus majorlate promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter,a MRP pol III promoter, a constitutive MPSV promoter, an RSV promoter, atetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and a constitutive CMV promoter.

The expression vectors should contain expression and replication signalscompatible with the cell in which the neukinase-encoding sequences areto be expressed. Expression vectors useful for expressingneukinase-encoding sequences include viral vectors such as retroviruses,adenoviruses and adenoassociated viruses, plasmid vectors, cosmids, andthe like. Viral and plasmid vectors are preferred for transfecting theexpression vectors into mammalian cells. For example, the expressionvector pcDNAI (Invitrogen, San Diego, Calif.), in which the expressioncontrol sequence comprises the CMV promoter, provides good rates oftransfection and expression into such cells.

The expression vectors can be introduced into the cell for expression ofthe neukinase-encoding sequence by any method known to one of skill inthe art without limitation. Such methods include, but are not limitedto, e.g., direct uptake of the recombinant DNA molecule by a cell fromsolution; facilitated uptake through lipofection using, e.g., liposomesor immunoliposomes; particle-mediated transfection; etc. See, e.g., U.S.Pat. No. 5,272,065; Goeddel et al., Methods in Fnzymology. vol. 185,Academic Press, Inc., CA (1990); Krieger, Gene Transfer and Expression—ALaboratory Manual, Stockton Press, New York (1990); Ausubel et al.,Current Protocols In Molecular Biology, John Wiley and Sons, New York(current edition); Sambrook et al., Molecular Cloning. A LaboratoryManual, 3d. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001).

The expression vectors can also contain a purification moiety thatsimplifies isolation of the expressed protein. For example, apolyhistidine moiety of, e.g., six histidine residues, can beincorporated at the amino terminal end of the protein. The polyhistidinemoiety allows convenient isolation of the protein in a single step bynickel-chelate chromatography. In certain embodiments, the purificationmoiety can be cleaved from the remainder of the delivery constructfollowing purification. In other embodiments, the moiety does notinterfere with the function of the functional domains of the expressedprotein and thus need not be cleaved.

5.3.3 Cells

In yet another aspect, the invention provides a cell comprising anexpression vector for expression of neukinase polypeptides of theinvention, or portions thereof. The cell is preferably selected for itsability to express high concentrations of the neukinase polypeptide tofacilitate subsequent purification of the polypeptide. In certainembodiments, the cell is a prokaryotic cell, for example, E. coli. In apreferred embodiment, the neukinase polypeptide is properly folded andcomprises the appropriate disulfide linkages when expressed in E. coli.

In other embodiments, the cell is a eukaryotic cell. Useful eukaryoticcells include yeast and mammalian cells. Any mammalian cell known by oneof skill in the art to be useful for expressing a recombinantpolypeptide, without limitation, can be used to express the deliveryconstructs. For example, Chinese hamster ovary (CHO) cells can be usedto express the neukinase polypeptides of the invention. In someembodiments, the neukinase polypeptide is expressed in neonatal ratventricular myocytes. In some embodiments, the neukinase polypeptide isexpressed in H9c2(2-1) cells.

5.4 Antibodies

According to the invention, neukinase, or its fragments thereof, may beused as an immunogen to generate antibodies which immunospecificallybind neukinase polypeptides. Such antibodies include, but are notlimited to, polyclonal, monoclonal, single chain monoclonal,recombinant, chimeric, humanized, mammalian, or human antibodies.

In some embodiments, antibodies to a non-human neukinase are produced.In certain embodiments, antibodies to rat neukinase are produced. Inother embodiments, antibodies to human neukinase are produced. Inanother embodiment, antibodies are produced that specifically bind to aprotein the amino acid sequence of which consists of SEQ ID NO: 1. Inanother embodiment, antibodies are produced that specifically bind to aprotein the amino acid sequence of which consists of SEQ ID NO:2. Inanother embodiment, antibodies are produced that specifically bind to aprotein the amino acid sequence of which consists of SEQ ID NO:25. Inanother embodiment, antibodies to a fragment of non-human neukinase areproduced. In another embodiment, antibodies to a fragment of ratneukinase are produced. In another embodiment, antibodies to a fragmentof human neukinase are produced. In a specific embodiment, fragments ofneukinase, human or non-human, identified as containing hydrophilicregions are used as immunogens for antibody production. In a specificembodiment, a hydrophilicity analysis can be used to identifyhydrophilic regions of neukinase, which are potential epitopes, and thuscan be used as immunogens.

For the production of antibody, various host animals can be immunized byinjection with native neukinase, or a synthetic version, or a fragmentthereof. In certain embodiments, the host animal is a mammal. In someembodiments, the mammal is a rabbit, mouse, rat, goat, cow or horse.

For the production of polyclonal antibodies to neukinase, variousprocedures known in the art may be used. In a particular embodiment,rabbit polyclonal antibodies to an epitope of neukinase encoded by asequence of SEQ ID NO:3 or SEQ ID NO:4 or a subsequence thereof, can beobtained. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species. Adjuvants that may be usedaccording to the present invention include, but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol. CpG-containing nucleic acids, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a neukinasepolypeptide, any technique that provides for the production of antibodymolecules by continuous cell lines in culture may be used. For example,monoclonal antibodies may be prepared by the hybridoma techniqueoriginally developed by Kohler and Milstein, Nature 256:495-497 (1975),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunol. Today 4:72 (1983)), or the EBV-hybridomatechnique (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)).

Techniques for the production of single chain antibodies, as describedin U.S. Pat. No. 4,946,778, can also be adapted to produce single chainantibodies specific to neukinase. An additional embodiment of theinvention utilizes the techniques described for the construction of Fabexpression libraries (Huse et al., Science 246:1275-1281 (1988)) toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for neukinase. Antibody fragments that contain theidiotype of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′),fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′), fragment, the Fab fragments which canbe generated by treating the antibody molecule with papain and areducing agent, and Fv fragments.

Techniques developed for the production of “chimeric” antibodies(Morrison et al., Proc. Natl. Acad. Sci. USA. 81:6851-6855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) can also be used. For example, nucleic acidsequences encoding a mouse antibody molecule specific to neukinase arespliced to nucleic acid sequences encoding a human antibody molecule.

In addition, techniques have been developed for the production ofhumanized antibodies, and such humanized antibodies to neukinase arewithin the scope of the present invention. See, e.g., Queen, U.S. Pat.No. 5,585,089 and Winter, U.S. Pat. No. 5,225,539. An immunoglobulinlight or heavy chain variable region consists of a “framework” regioninterrupted by three hypervariable regions, referred to ascomplementarity determining regions (CDRs). The extent of the frameworkregion and CDRs have been precisely defined. See, Sequences of Proteinsof Immunological Interest, Kabat, E. et al., U.S. Department of Healthand Human Services (1983). Briefly, humanized antibodies are antibodymolecules from non-human species having one or more CDRs from thenon-human species and a framework region from a human immunoglobulinmolecule.

Human antibodies may be used and can be obtained by using humanhybridomas (Cote et al., Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030(1983)) or by transforming human B cells with EBV virus in vitro (Coleet al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp.77-96 (1985)).

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or RIBA(recombinant immunoblot assay). For example, to select antibodies whichrecognize a specific domain of neukinase, one may assay generatedhybridomas for a product which binds to a neukinase fragment containingsuch domain. For selection of an antibody that specifically binds afirst neukinase homologue but which does not specifically bind a second,different neukinase homologue, one can select on the basis of positivebinding to the first neukinase homologue and a lack of binding to thesecond neukinase homologue.

Antibodies specific to a domain of neukinase or a homologue thereof arealso provided. The foregoing antibodies can be used in methods known inthe art relating to the localization and activity of the neukinase ofthe invention, e.g., for imaging these proteins, measuring levelsthereof in appropriate physiological samples, in diagnostic methods,etc.

5.5 Transgenic Neukinase Animals

Transgenic animals are useful for studying the function and/or activityof neukinase and for identifying and/or evaluating modulators ofneukinase activity. Transgenes direct the expression of an encoded geneproduct in one or more cell types or tissues of the transgenic animal.In some embodiments, transgenes prevent the expression of a naturallyencoded gene product in one or more cell types or tissues (a “knockout”transgenic animal). In some embodiments, transgenes serve as a marker orindicator of an integration, chromosomal location, or region ofrecombination (e.g., cre/loxP mice).

A transgenic animal can be created by introducing a nucleic acid of theinvention into the male pronuclei of a fertilized oocyte (e.g., bymicroinjection, retroviral infection) and allowing the oocyte to developin a pseudopregnant female foster animal (PFFA). The neukinase sequencescan be introduced as a transgene into the genome of a non-human animal.In some embodiments, the neukinase sequence is the rat neukinasesequence (SEQ ID NO:3). In some embodiments, the neukinase sequence isthe human neukinase sequence (SEQ ID NO:4). In other embodiments, ahomologue of neukinase can be used as a transgene. Intronic sequencesand polyadenylation signals can also be included in the transgene toincrease transgene expression. Tissue-specific regulatory sequences canbe operably-linked to the neukinase transgene to direct expression ofneukinase to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art, e.g, Evans et al., U.S. Pat.No. 4,870,009 (1994): Leder and Stewart, U.S. Pat. No. 4,736,866, 1988;Wagner and Hoppe, U.S. Pat. No. 4,873,191 (1989). Other non-micetransgenic animals may be made by similar methods. A transgenic founderanimal, which can be used to breed additional transgenic animals, can beidentified based upon the presence of the transgene in its genome and/orexpression of the transgene mRNA in tissues or cells of the animal.Transgenic neukinase animals can be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector containing at leasta portion of neukinase into which a deletion, addition or substitutionmay be introduced to thereby alter, e.g., functionally disrupt neukinaseexpression. In some embodiments, the vector may contain a neomycincassette inserted in reverse orientation relative to neukinasetranscription to functionally disrupt neukinase. Neukinase can be ahuman gene (SEQ ID NO: 10), or other neukinase homologue. In oneapproach, a knockout vector functionally disrupts the endogenousneukinase gene upon homologous recombination, and thus a non-functionalneukinase protein, if any, is expressed.

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous neukinase is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of endogenousneukinase). In this type of homologous recombination vector, the alteredportion of the neukinase sequence is flanked at its 5′- and 3′-terminiby additional nucleic acid sequence of neukinase to allow for homologousrecombination to occur between the exogenous neukinase sequence carriedby the vector and an endogenous neukinase sequence in an embryonic stemcell. The additional flanking neukinase sequence is sufficient toengender homologous recombination with endogenous neukinase. Typically,several kilobases of flanking DNA (both at the 5′- and 3′-termini) areincluded in the vector (see Thomas and Capecchi, Cell 51:503-512(1987)).

The vector is then introduced into an embryonic stem cell line (e.g., byelectroporation), and cells in which the introduced neukinase sequencehas homologously-recombined with the endogenous neukinase sequence areselected (Li et al., Cell 69:915-926 (1992)).

Selected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see Bradley, Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Oxford University Press,Inc., Oxford (1987)). A chimeric embryo can then be implanted into asuitable PFFA, wherein the embryo is brought to term. Progeny harboringthe homologously-recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain thehomologously-recombined DNA by germline transmission of the transgene.Methods for constructing homologous recombination vectors and homologousrecombinant animals are described (Berns et al., WO 93/04169, 1993;Kucherlapati et al., WO 91/01140, 1991; Le Mouellic and Brullet, WO90/11354, 1990).

Alternatively, transgenic animals that contain selected systems thatallow for regulated expression of the transgene can be produced. Anexample of such a system is the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al., Proc. Natl. Acad. Sci. USA 89:6232-6236(1992)). Another recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al., Science 251:1351-1355(1991)). If a cre/loxP recombinase system is used to regulate expressionof the transgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beproduced as “double” transgenic animals, by mating an animal containinga transgene encoding a selected protein to another containing atransgene encoding a recombinase.

Clones of transgenic animals can also be produced (Wilmut et al., Nature385:810-813 (1997)). In brief, a cell from a transgenic animal can beisolated and induced to exit the growth cycle and enter Go phase. Thequiescent cell can then be fused to an enucleated oocyte from an animalof the same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured to develop to a morula orblastocyte and then transferred to a PFFA. The offspring borne of thisfemale foster animal will be a clone of the “parent” transgenic animal.

5.6 Methods of Screenine for Modulators of Neukinase Activity

The present invention also provides methods of identifying a compoundthat modulates the activity of neukinase in a cell or tissue ofinterest. A compound may modulate neukinase activity by affecting, forexample: (1) the number of copies of the neukinase gene in the cell(amplifiers and deamplifiers); (2) increasing or decreasingtranscription of the neukinase gene (transcription up-regulators anddown-regulators); (3) by increasing or decreasing the translation of theneukinase mRNA into protein (translation up regulators and downregulators); or (4) by increasing or decreasing the activity of theneukinase protein (agonists and antagonists). To identify compounds thataffect neukinase at the DNA, RNA, and protein levels, cells or organismsare contacted with a candidate compound and the corresponding change inneukinase DNA, RNA or protein may be assessed. For DNA amplifiers ordeamplifiers, the amount of neukinase DNA may be measured. For thosecompounds that are transcription up-regulators and down-regulators, theamount of neukinase mRNA may be measured. Alternatively, the neukinasepromoter sequence may be operably linked to a reporter gene, andpotential transcriptional modulators of neukinase may be assayed bymeasuring reporter gene activity in the presence and absence of thecompound. For translational up- and down-regulators, the amount ofneukinase polypeptide may be measured. Alternatively, changes inneukinase biological activity, as measured by the techniques describedbelow, may be an indirect indicator of the ability of a compound tomodulate neukinase translation.

Neukinase activity of the methods described herein encompasses thebiological activity of neukinase, which includes, but is not limited to,phosphorylation of cardiac myosin light chain and/or functionalfragments or variants of myosin light chain, calmodulin binding, andautoinhibition. Methods for examining cell-based phosphorylation eventsare commonly known in the art, and may be utilized to examine changes inmyosin light chain phosphorylation following contact with a putativemodulator of neukinase biological activity.

In one embodiment, the cell or tissue useful for the methods describedherein expresses a neukinase polypeptide from an endogenous copy of theneukinase gene. In another embodiment, the cell or tissue expresses aneukinase polypeptide following transient or stable transformation witha nucleic acid encoding a neukinase polypeptide of the presentinvention. Any mammalian cell known by one of skill in the art to beuseful for expressing a recombinant polypeptide, without limitation, canbe used to express a neukinase polypeptide useful for the methodsdescribed herein.

In one embodiment, the method of identifying a compound that modulatesthe activity of neukinase comprises determining a first level ofneukinase activity in a cell or tissue that expresses a neukinasepolypeptide, contacting said cell or tissue with a test compound, thendetermining a second level of neukinase activity in said cell or tissue.A difference in the first level and second level of neukinase activityis indicative of the ability of the test compound to modulate neukinaseactivity. In one embodiment, a compound may have agonistic activity ifthe second level of neukinase activity is greater than the first levelof neukinase activity. In certain embodiments, agonistic activitycomprises at least about a 2, 4, 6, 8, 10, or greater fold increase inthe second level of neukinase activity compared to the first level ofneukinase activity. In another embodiment, a compound may haveantagonistic activity if the second level of neukinase activity is lessthan the first level of neukinase activity. In certain embodiments,antagonistic activity comprises at least about a 2, 4, 6, 8, 10, orgreater fold decrease in the second level of neukinase activity comparedto the first level of neukinase activity.

In another embodiment, the invention provides a method of identifying acompound that modulates the activity of neukinase in a cell or tissueexpressing a neukinase polypeptide, comprising contacting said cell ortissue with a test compound and determining a level of neukinase in saidcell or tissue. The difference in this level and a standard or baselinelevel of neukinase activity in a comparable cell or tissue, e.g., acontrol cell or tissue not contacted with the test compound, isindicative of the ability of said test compound to modulate neukinaseactivity. In one embodiment, a compound may have agonistic activity ifthe level of neukinase activity in the cell or tissue contacted withsaid compound is greater than the level of neukinase activity in thecontrol cell or tissue. In certain embodiments, agonistic activitycomprises at least about a 2-, 4-, 6-, 8-, 10-, or greater fold increasein the level of neukinase activity of a cell or tissue contacted withthe test compound compared to the level of neukinase activity in thecontrol cell or tissue. In another embodiment, a compound may haveantagonistic activity if the level of neukinase activity in the cell ortissue contacted with said compound is less than the level of neukinaseactivity in the control cell or tissue. In certain embodiments,antagonistic activity comprises at least about a 2-, 4-, 6-, 8-, 10-, orgreater fold decrease in the level of neukinase activity of a cell ortissue contacted with the test compound compared to the level ofneukinase activity in the control cell or tissue.

The present invention also provides methods of identifying a compoundthat modulates the activity of neukinase in a transgenic non-humananimal which expresses a neukinase polypeptide, comprising administeringthe compound to said animal and assessing the animal for an alterationin cardiac function affected by the compound. Cardiac function may beassessed through the measurement of interventricular septum size, leftventricle end diastolic dimension (LVEDD), posterior wall thickness,left ventricle end systolic dimension (LVESD), ejection fraction (EF),fractional shortening (FS), and cardiac cycle. In one embodiment, acompound may have agonistic activity if the LVEDD value followingadministration of the compound is reduced by at least about 2%, 5%, 10%,15%, 20%, or greater. In another embodiment, a compound may haveagonistic activity if the LVESD value following administration of thecompound is reduced by at least about 2%, 5%, 10%, 15%, 20%, or greater.In another embodiment, a compound may have agonistic activity if the EFvalue of the left ventricle is enhanced by at least about 10%, 20%, 30%,40%, 50%, 60% or greater. In another embodiment, a compound may haveagonistic activity if the FS value of the left ventricle is enhanced byat least about 10%, 20%, 30%, 40%, 50%, 60% or greater.

The present invention also provides methods of identifying compoundsthat specifically bind to neukinase nucleic acids or polypeptides andthus have potential use as agonists or antagonists of neukinase. Incertain embodiments, such compounds may affect cardiac hypertrophy,ventricular muscle cell hypertrophy, etc. In a preferred embodiment,assays are performed to screen for compounds having potential utility asheart failure therapies or lead compounds for drug development. Theinvention thus provides assays to detect compounds that specificallybind to neukinase nucleic acids or polypeptides. For example,recombinant cells expressing neukinase nucleic acids can be used torecombinantly produce neukinase polypeptides for use in these assays,e.g., to screen for compounds that bind to neukinase polypeptides. Saidcompounds (e.g., putative binding partners of neukinase) are contactedwith a neukinase polypeptide or a fragment thereof under conditionsconducive to binding, and compounds that specifically bind to neukinaseare identified. Similar methods can be used to screen for compounds thatbind to neukinase nucleic acids. Methods that can be used to carry outthe foregoing are commonly known in the art.

In some embodiments, cell free assays utilizing a purified neukinasepolypeptide may be performed to identify compounds which modulate (1)the phosphorylation of cardiac myosin light chain and/or functionalfragments or variants thereof, (2) the autoinhibitory activity ofneukinase in the absence of Ca²⁺/calmodulin, and/or (3) neukinasebinding of, and activation by, calmodulin. Myosin light chain kinaseassays are well known in the art, and are described, for example, byPolak et al., J. Neurosci. 11:534-54 (1991), Ausubel et al., CurrentProtocols In Molecular Biology, John Wiley and Sons, New York (currentedition), and U.S. Pat. No. 5,906,810, the contents of which are herebyincorporated by reference in their entirety. Putative modulators ofneukinase biological activity may be identified by assaying neukinasekinase activity in the presence of varying concentrations of thecompound and examining the extent of phosphate incorporation into asuitable substrate. In some embodiments, the substrate is myosin lightchain. In some embodiments, the substrate is a functional fragment ofmyosin light chain. In some embodiments, the substrate is a variant ofmyosin light chain.

In certain embodiments, modulation of neukinase activity may be measuredby calmodulin activity assays, as described in U.S. Pat. No. 5,840,697,Sharma et al., Adv. Cyclic Nucleotide Res. 10:187-89 (1979), and Wallaceet al., Methods Enzymol., 102:39-47 (1983), the contents of which areherein incorporated by reference in their entireties. Compounds whichbind to and inhibit calmodulin activity may also inhibit Ca²⁺/calmodulindependent activation of neukinase. By way of example and not limitation,calmodulin activity in the presence and absence of potential modulatorsof neukinase activity may be measured using a calcium dependentphosphodiesterase assay. Calmodulin activity is measured by its abilityto stimulate phosphodiesterase activity as determined by a two-stepassay procedure illustrated by reactions (1) and (2) below.

During the first step of the assay, cyclic adenosine 3′5′-monophosphate(cAMP) is incubated with calcium-activated phosphodiesterase (Cal-PDE),which hydrolyses the 3′bond producing adenosine 5′-monophosphate(5′-AMP). During the second step, 5′-AMP is quantitatively convertedinto adenosine and inorganic phosphate (Pi) through the action of a5-nucleotidase. The reaction is followed by the measurement of the Piformed by reading the absorbance at 660 nm after reacting with ammoniummolybdate. The amount of Pi formed is directly related to thephosphodiesterase activity which depends on the level of activation bycalmodulin.

In various embodiments, the neukinase-modulating compound is a protein,for example, an antibody; a nucleic acid; or a small molecule. As usedherein, the term “small molecule” includes, but is not limited to,organic or inorganic compounds (i.e., including heteroorganic andorganometallic compounds) having a molecular weight less than 10,000grams per mole, organic or inorganic compounds having a molecular weightless than 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than 1,000 grams per mole, organic or inorganiccompounds having a molecular weight less than 500 grams per mole,organic or inorganic compounds having a molecular weight less than 100grams per mole, and salts, esters, and other pharmaceutically acceptableforms of such compounds. Salts, esters, and other pharmaceuticallyacceptable forms of such compounds are also encompassed.

By way of example, diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries can be screened for molecules thatspecifically bind to neukinase. Many libraries are known in the art thatcan be used, e.g., chemically synthesized libraries, recombinant (e.g.,phage display libraries), and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal., Science 251:767-773 (1991); Houghten et al., Nature 354:84-86(1991); Lam et al., Nature 354:82-84 (1991): Medynski, Bio/Technology12:709-710 (1994); Gallop et al., J. Medicinal Chemistry 37(9):1233-1251(1994); Ohlmeyer et al., Proc. Natl. Acad. Sci. U.S.A. 90:10922-10926(1993): Erb et al., Proc. Natl. Acad. Sci. U.S.A. 91:11422-11426 (1994):Houghten et al., Biotechniques 13:412 (1992); Jayawickreme et al., Proc.Natl. Acad Sci. U.S.A. 91:1614-1618 (1994); Salmon et al., Proc. Natl.Acad. Sci. U.S.A. 90:11708-11712 (1993): PCT Publication No. WO93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. U.S.A.89:5381-5383 (1992).

Examples of phage display libraries are described in Scott and Smith.Science 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990);Christian. R. B., et al., J. Mol. Biol. 227:711-718 (1992)); Lenstra, J.Immunol. Meth. 152:149-157 (1992); Kay et al., Gene 128:59-65 (1993);and PCT Publication No. WO 94/18318, published Aug. 18, 1994. In vitrotranslation-based libraries include but are not limited to thosedescribed in PCT Publication No. WO 91/05058, published Apr. 18, 1991;and Mattheakis et al., Proc. Natl. Acad. Sci. U.S.A. 91:9022-9026(1994).

By way of examples of non-peptide libraries, a benzodiazepine library(see e.g., Bunin et al., Proc. Natl. Acad. Sci. U.S.A. 91:4708-4712(1994)) can be adapted for use. Peptoid libraries (Simon et al., Proc.Natl. Acad. Sci. U.S.A. 89:9367-9371 (1992)) can also be used. Anotherexample of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal., Proc. Natl. Acad. Sci. U.S.A. 91:11138-11142 (1994).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, Adv. Exp.Med. Biol. 251:215-218 (1989): Scott and Smith, Science 249:386-390(1990): Fowlkes et al., Bio/Techniques 13:422-427 (1992); Oldenburg etal., Proc. Natl. Acad. Sci. U.S.A. 89:5393-5397 (1992); Yu et al., Cell76:933-945 (1994); Staudt et al., Science 241:577-580 (1988); Bock etal., Nature 355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci.U.S.A. 89:6988-6992 (1992): Ellington et al., Nature 355:850-852 (1992);U.S. Pat. No. 5,096,815. U.S. Pat. No. 5,223,409, and U.S. Pat. No.5,198,346; Rebar and Pabo, Science 263:671-673 (1993); and PCTPublication No. WO 94/18318, published Aug. 8, 1994.

In a specific embodiment, screening can be carried out by contacting thelibrary members with neukinase polypeptide (or nucleic acid) immobilizedon a solid phase and harvesting those library members that bind to theprotein (or nucleic acid). Examples of such screening methods, termed“panning” techniques are described by way of example in Parmley andSmith, Gene 73:305-318 (1988): Fowlkes et al., Bio/Techniques 13:422-427(1992); PCT Publication No. WO 94/18318; and in references cited hereinabove.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, Nature 340:245-246 (1989); Chien etal., Proc. Natl. Acad. Sci. U.S.A. 88:9578-9582 (1991)) can be used toidentify molecules that specifically bind to neukinase protein or ananalog thereof.

In another embodiment, screening can be carried out by creating apeptide library in a prokaryotic or eukaryotic cell, such that thelibrary proteins are expressed on the cells' surface, followed bycontacting the cell surface with neukinase and determining whetherbinding has taken place. Alternatively, the cells are transformed with anucleic acid encoding neukinase, such that neukinase is expressed on thecells' surface. The cells are then contacted with a potential agonist orantagonist, and binding, or lack thereof, is determined. In a specificembodiment of the foregoing, the potential agonist or antagonist isexpressed in the same or a different cell such that the potentialagonist or antagonist is expressed on the cells' surface.

As would clearly be understood by a person of ordinary skill in the art,any and/or all of the embodiments disclosed herein for identifying anagent, drug, or compound that can modulate the activity of neukinase,including such procedures that incorporate rational drug design, asdisclosed herein, can be combined to form additional drug screens andassays, all of which are contemplated by the present invention.

5.7 Diagnostic Methods

The present invention also pertains to the field of predictive medicinein which diagnostic and prognostic assays are used for prognostic(predictive) purposes to treat an individual prophylactically.Accordingly, one aspect of the invention relates to diagnostic assaysfor determining neukinase nucleic acid expression as well as neukinaseactivity in the context of a biological sample (e.g., blood, serum,cells, tissue) to determine whether an individual is afflicted with adisease or disorder, or is at risk of developing a disorder. Such adisease or disorder may be associated with aberrant neukinase expressionor activity, and can include, but is not limited to, cardiacdysfunction. In particular embodiments, the cardiac dysfunction ishypertrophic cardiomyopathy. In other embodiments, the cardiacdysfunction is heart failure. The invention also provides for prognosticassays for determining whether an individual is at risk of developing adisorder associated with neukinase nucleic acid expression or activity.For example, mutations in neukinase can be assayed in a biologicalsample. Such assays can be used for prognostic or predictive purpose toprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with aberrant neukinase nucleic acidexpression or biological activity.

5.7.1 Diagnostic Assays

An exemplary method for detecting the presence or absence of neukinasein a biological sample involves obtaining a biological sample from asubject and contacting the biological sample with a compound or an agentcapable of detecting neukinase nucleic acid (e.g., mRNA, genomic DNA)such that the presence of neukinase is confirmed in the sample. An agentfor detecting neukinase mRNA or genomic DNA is a labeled nucleic acidprobe that can hybridize to neukinase mRNA or genomic DNA. The nucleicacid probe can be, for example, a full-length neukinase nucleic acid,such as the nucleic acid of SEQ ID NOS:3 or 4, or a portion thereof. Insome embodiments, the nucleic acid probe is an oligonucleotide of atleast 15, 30, 50, 100, 250 or 500 nucleotides in length and issufficient to specifically hybridize under stringent conditions toneukinase mRNA or genomic DNA.

An agent for detecting neukinase polypeptide can be an antibody capableof binding to neukinase, preferably an antibody with a detectable label.Antibodies can be polyclonal or monoclonal. An intact antibody or anantibody fragment, e.g., a Fab fragment, can be used. A labeled probe orantibody may be coupled (i.e., physically linked) to a detectablesubstance, or an indirect detection method may be employed wherein theprobe or antibody is detected via reactivity with a directly labeledsecondary reagent. Examples of indirect labeling include detection of aprimary antibody using a fluorescently labeled secondary antibody, orend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin.

The detection method of the invention can be used to detect neukinasemRNA, protein, or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of neukinasemRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of neukinase polypeptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of neukinase genomic DNA include Southern hybridizations andfluorescence in situ hybridization (FISH). Furthermore, in vivotechniques for detecting neukinase include introducing into a subject alabeled anti-neukinase antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In one embodiment, the biological sample from the subject containsprotein molecules, and/or mRNA molecules, and/or genomic DNA molecules.In certain embodiments, the biological sample is blood.

In another embodiment, the methods further involve obtaining abiological sample from a subject to provide a control, contacting thesample with a compound or agent to detect neukinase mRNA or genomic DNA,and comparing the presence of neukinase mRNA or genomic DNA in thecontrol sample with the presence of neukinase mRNA or genomic DNA in thetest sample.

5.7.2 Prognostic Assays

The diagnostic methods described herein can be further utilized toidentify subjects having, or who are at risk of developing, a disease ordisorder associated with aberrant neukinase expression or activity. Sucha disease or disorder may include, but is not limited to, cardiacdysfunction, particularly hypertrophic cardiomyopathy and heart failure.The invention provides a method for identifying a disease or disorderassociated with aberrant neukinase expression or activity in which atest sample is obtained from a subject and neukinase nucleic acid (e.g.,mRNA, genomic DNA) is detected. A test sample is a biological sampleobtained from a subject. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Prognostic assays can be used to determine whether a subject can beadministered a modality (e.g., an agonist, antagonist, peptidomimetic,protein, peptide, nucleic acid, small molecule, food, etc.) to treat adisease or disorder associated with aberrant neukinase expression oractivity. Such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder. The invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a disorder associated with aberrant neukinaseexpression or activity in which a test sample is obtained and neukinasenucleic acid is detected (e.g., where the presence of neukinase nucleicacid is diagnostic for a subject that can be administered the agent totreat a disorder associated with aberrant neukinase expression oractivity).

The methods of the invention can also be used to detect genetic lesionsin a neukinase gene to determine if a subject with the genetic lesion isat risk for a disorder, including but not limited to hypertrophiccardiomyopathy or heart failure. Methods include detecting, in a samplefrom the subject, the presence or absence of a genetic lesioncharacterized by an alteration affecting the integrity of a geneencoding a neukinase polypeptide, or the mis-expression of a neukinasegene. Such genetic lesions can be detected by ascertaining: (1) adeletion of one or more nucleotides from the neukinase gene; (2) anaddition of one or more nucleotides to the neukinase gene; (3) asubstitution of one or more nucleotides in the neukinase gene; (4) achromosomal rearrangement of a neukinase gene; (5) an alteration in thelevel of neukinase mRNA transcripts; (6) aberrant modification of aneukinase gene, such as a change in genomic DNA methylation; (7) thepresence of a non-wild-type splicing pattern of a neukinase mRNAtranscript, (8) a non-wild-type level of a neukinase polypeptide; (9)allelic loss of neukinase; and/or (10) inappropriate post-translationalmodification of a neukinase polypeptide. There are a large number ofknown assay techniques that can be used to detect lesions in neukinase.Any biological sample containing nucleated cells may be used.

Detection of genetic lesions of neukinase may employ any technique knownin the art. In certain embodiments, lesion detection may employ anucleic acid probe/primer in a polymerase chain reaction (PCR) reactionsuch as anchor PCR or rapid amplification of cDNA ends (RACE) PCR. Thismethod may include collecting a sample from a patient, isolating nucleicacids from the sample, contacting the nucleic acids with one or morenucleic acid primers that specifically hybridize to neukinase nucleicacid under conditions such that hybridization and amplification of theneukinase sequence (if present) occurs, and detecting the presence orabsence of an amplification product, or detecting the size of theamplification product and comparing the length to a control sample. Itis anticipated that PCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Mutations in a neukinase gene from a sample can also be identified byalterations in restriction enzyme cleavage patterns. For example, sampleand control DNA is isolated, amplified (optionally), digested with oneor more restriction endonucleases, and fragment length sizes aredetermined by gel electrophoresis and compared. Differences in fragmentlength sizes between sample and control DNA indicate mutations in thesample DNA. Moreover, the use of sequence specific ribozymes can be usedto score for the presence of specific mutations by development or lossof a ribozyme cleavage site.

Furthermore, hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high-density arrays containing hundreds or thousands ofoligonucleotides probes can identify genetic mutations in neukinase (seeCronin et al., Hum. Mutat. 7:244-255 (1996); Kozal et al., Nat. Med.2:753-759 (1996)). For example, genetic mutations in neukinase can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin, et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This is followed by asecond hybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the neukinase gene anddetect mutations by comparing the sequence of the sample neukinasesequence with the corresponding wild-type (control) sequence. Examplesof sequencing reactions include those based on classic techniques (seeMaxam and Gilbert, Proc. Natl. Acad Sci USA 74:560-564 (1977); Sanger etal., Natl. Acad Sci USA 74:5463-5367 (1977)). Any of a variety ofautomated sequencing procedures can be used for performing diagnosticassays of the present invention (see Naeve et al., Biotechniques19:448-453 (1995)) including sequencing by mass spectrometry (Cohen etal., Adv. Chromatogr. 36:127-162 (1996), Griffin and Griffin. Appl.Biochem. Biotechnol. 38:147-159 (1993)).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (see Saiki et al.,Nature 324:163-166 (1986); Saiki et al., Proc. Natl. Acad Sci. USA86:6230-6234 (1989)). Such allele-specific oligonucleotides arehybridized to PCR-amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

5.8 Compositions

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a therapeutic ofthe invention. In a preferred aspect, the therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably human. In a specificembodiment, a non-human mammal is the subject. Formulations and methodsof administration that can be employed can be selected from among thosedescribed herein below.

Various delivery systems are known and can be used to administer atherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a therapeutic nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommavareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.

In another embodiment, the therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-372, 353-365(1989)).

In yet another embodiment, the therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Seflon, CRC Crit. Ref Biomed. Eng. 14:201 (1987):Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974): Controlled Drug Bioavailability:Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Pewas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the thymus, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the therapeutic is a nucleic acidencoding a protein therapeutic (e.g., SEQ ID NO: 1 or SEQ ID NO:2), thenucleic acid can be administered in vivo to promote expression of itsencoded protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic. DuPont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad Sci. U.S.A.88:1864-1868 (1991)), etc. Alternatively, a nucleic acid therapeutic canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of atherapeutic, and a pharmaceutically acceptable carrier. Water is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences by E. W. Martin.Such compositions will contain a therapeutically effective amount of thetherapeutic, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

5.9 Kits

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration. Whenthe invention is supplied as a kit, the different components of thecomposition may be packaged in separate containers and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

Kits may also include reagents in separate containers that facilitatethe execution of a specific test, such as diagnostic tests or tissuetyping. For example, neukinase DNA templates and suitable primers may besupplied for internal controls.

5.9.1 Containers or Vessels

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved, andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized luciferase orbuffer that have been packaged under a neutral, non-reacting gas, suchas nitrogen. Ampoules may consist of any suitable material, such asglass, organic polymers, such as polycarbonate, polystyrene, etc.,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include simple bottlesthat may be fabricated from similar substances as ampules, andenvelopes, that may consist of foil-lined interiors, such as aluminum oran alloy. Other containers include test tubes, vials, flasks, bottles,syringes, or the like. Containers may have a sterile access port, suchas a bottle having a stopper that can be pierced by a hypodermicinjection needle. Other containers may have two compartments that areseparated by a readily removable membrane that upon removal permits thecomponents to mix. Removable membranes may be glass, plastic, rubber,etc.

5.9.2 Instructional Materials

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

5.10 Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk for (or susceptible to) a disorder or havinga disorder associated with aberrant neukinase expression or activity.Exemplary disorders are characterized by abnormal cardiac function,including, but not limited to, congestive heart failure, myocardialinfarction, tachyarrythmia, familial cardiac hypertrophy, ischemic heartdisease, idiopathic dilated cardiomyopathy, myocarditis and the like.

5.10.1 Diseases and Disorders

Diseases and disorders that are characterized by increased neukinaselevels or biological activity may be treated with therapeutics thatantagonize (i.e., reduce or inhibit) activity. Antagonists may beadministered in a therapeutic or prophylactic manner. Therapeutics thatmay be used include: (1) neukinase peptides, or analogs, derivatives,fragments or homologues thereof; (2) Abs to a neukinase peptide; (3)neukinase nucleic acids; (4) administration of antisense nucleic acidand nucleic acids that are “dysfunctional” (i.e., due to a heterologousinsertion within the coding sequences) that are used to eliminateendogenous function of neukinase by homologous recombination (Capecchi,Science 244:1288-1292 (1989)); or (5) modulators (i.e., inhibitors,agonists and antagonists, including additional peptide mimetic of theinvention or Abs specific to neukinase) that alter the interactionbetween neukinase and its binding partner.

Diseases and disorders that are characterized by decreased neukinaselevels or biological activity may be treated with therapeutics thatincrease (i.e., are agonists to) activity. Therapeutics that upregulateactivity may be administered therapeutically or prophylactically.Therapeutics that may be used include peptides, or analogs, derivatives,fragments or homologues thereof; or an agonist that increasesbioavailability, or, in a specific embodiment, an agonist that increasesneukinase activity by inhibiting the autoinhibitory domain of neukinase.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or neukinasemRNAs). Methods include, but are not limited to, immunoassays (e.g., byWestern blot analysis, immunoprecipitation followed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry,etc.) and/or hybridization assays to detect expression of mRNAs (e.g.,Northern assays, dot blots, in situ hybridization, and the like).

5.10.2 Prophylactic Methods

The invention provides a method for preventing, in a subject, a diseaseor condition associated with an aberrant neukinase expression oractivity, by administering an agent that modulates neukinase expressionor at least one neukinase activity. Subjects at risk for a disease thatis caused or contributed to by aberrant neukinase expression or activitycan be identified by, for example, any or a combination of diagnostic orprognostic assays. Administration of a prophylactic agent can occurprior to the manifestation of symptoms characteristic of the neukinaseaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. In a specific embodiment ofthe invention, ventricular muscle cell hypertrophy is prevented ordelayed by administration of said prophylactic agent. Depending on thetype of neukinase aberrancy, for example, a neukinase agonist orneukinase antagonist can be used to treat the subject. The appropriateagent can be determined based on screening assays.

5.10.3 Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingneukinase expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of neukinase activityassociated with the cell. An agent that modulates neukinase activity canbe a nucleic acid or a protein, a naturally occurring cognate ligand ofneukinase, a peptide, a neukinase peptidomimetic, an aptamer, or othersmall molecule. The agent may stimulate neukinase activity. Examples ofsuch stimulatory agents include active neukinase and a neukinase nucleicacid molecule that has been introduced into the cell. Stimulation ofneukinase activity is desirable in situations in which neukinase isabnormally down-regulated and/or in which increased neukinase activityis likely to have a beneficial effect.

In other embodiments, the neukinase-modulating agent inhibits neukinaseactivity. Examples of inhibitory agents include anti-neukinase Abs, oran inhibitory nucleic acid molecule. For example, the nucleic acidmolecule may comprise an antisense oligonucleotide, an aptamer, or aninhibitory/interfering RNA (e.g., a small inhibitory/interfering RNA.Methods for screening for, identifying and making these nucleic acidmodulators are known in the art.

In some embodiments, RNA interference (RNAi) (see, e.g. Chuang et al.,Proc. Natl. Acad Sci. U.S.A. 97:4985 (2000)) can be employed to inhibitthe expression of a gene encoding neukinase. Interfering RNA (RNAi)fragments, particularly double-stranded (ds) RNAi, can be used togenerate loss-of-neukinase function. Methods relating to the use of RNAito silence genes in organisms, including mammals, C. elegans,Drosophila, plants, and humans are known (see, e.g., Fire et al., Nature391:806-811 (1998); Fire, Trends Genet. 15:358-363 (1999); Sharp, GenesDev. 15:485-490 (2001); Hammond, et al., Nature Rev. Genet. 2:1110-1119(2001): Tuschl, Chem. Biochem. 2:239-245 (2001); Hamilton et al.,Science 286:950-952 (1999); Hammond et al., Nature 404:293-296 (2000):Zamore et al., Cell 101:25-33 (2000); Bernstein et al., Nature 409:363-366 (2001): Elbashir et al., Genes Dev. 15:188 200 (2001); Elbashiret al. Nature 411:494-498 (2001); International PCT application No. WO01/29058; and International PCT application No. WO 99/32619), thecontents of which are incorporated by reference. Double-stranded RNA(dsRNA)-expressing constructs are introduced into a host using areplicable vector that remains episomal or integrates into the genome.By selecting appropriate sequences, expression of dsRNA can interferewith accumulation of endogenous mRNA encoding neukinase.

Modulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe agent to a subject). As such, the invention provides methods oftreating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a neukinase ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assay),or combination of agents that modulates (e.g., up-regulates ordown-regulates) neukinase expression or activity. In another embodiment,the method involves administering a neukinase or nucleic acid moleculeas therapy to compensate for reduced or aberrant neukinase expression oractivity.

5.10.4 Determination of the Biological Effect of the Therapeutic

Suitable in vitro or in vive assays can be performed to determine theeffect of a specific therapeutic and whether its administration isindicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays may be performed withrepresentative cells of the type(s) involved in the patient's disorder,to determine if a given therapeutic exerts the desired effect upon thecell type(s). Modalities for use in therapy may be tested in suitableanimal model systems including, but not limited to rats, mice, chicken,cows, monkeys, rabbits, and the like, prior to testing in humansubjects.

Similarly, for in vivo testing, any of the animal model systems known inthe art may be used prior to administration to human subjects. In oneembodiment, a candidate therapeutic may be tested for efficacy in an invivo model for cardiac hypertrophy. The in vivo determination ofhypertrophy includes measurement of cardiovascular parameters such asblood pressure, heart rate, systemic vascular resistance, contractility,force of heart beat, concentric or dilated hypertrophy, left ventricularsystolic pressure, left ventricular mean pressure, left ventricularend-diastolic pressure, cardiac output, stroke index, histologicalparameters, and ventricular size and wall thickness. Animal modelsavailable for determination of development and suppression ofventricular muscle cell hypertrophy in vivo include thepressure-overload mouse model, RV murine dysfunctional model, transgenicmouse model, and post-myocardial infarction rat model. Medical methodsfor assessing the presence, development, and suppression of ventricularmuscle cell hypertrophy in human patients are known, and include, forexample, measurements of diastolic and systolic parameters, estimates ofventricular mass, and pulmonary vein flows.

5.10.5 Prophylactic and Therapeutic Uses of the Compositions of theInvention

Neukinase nucleic acids and proteins are useful in potentialprophylactic and therapeutic applications implicated in a variety ofdisorders including, but not limited to, congestive heart failure,myocardial infarction, tachyarrythmia, familial cardiac hypertrophy,ischemic heart disease, idiopathic dilated cardiomyopathy, myocarditisand the like.

As an example, a cDNA encoding neukinase may be useful in gene therapy,and the protein may be useful when administered to a subject in needthereof. By way of non-limiting example, the compositions of theinvention will have efficacy for treatment of patients suffering fromheart failure.

Neukinase nucleic acids, or fragments thereof, may also be useful indiagnostic applications, wherein the presence or amount of the nucleicacid or the protein is to be assessed. A further use could be as ananti-bacterial molecule (i.e., some peptides have been found to possessanti-bacterial properties). These materials are further useful in thegeneration of Abs that immunospecifically bind to the novel substancesof the invention for use in therapeutic or diagnostic methods.

6. EXAMPLES

The invention is illustrated by the following examples which are notintended to be limiting in any way.

6.1 Example 1 Upregulation of Neukinase Gene Expression FollowingApplication of Neuregulin in Myocardial Infarcted Rat Left Ventricle

In order to identify genes which may be regulated by neuregulin (NRG),gene expression was examined in both normal and myocaridal infarcted ratleft ventricle following extended infusion of NRG by osmotic pump.

To charge an osmotic pump with NRG, 1 ml of sterile water and 1 ml ofsterile 0.9% saline was injected into a vial of NRG (993.1 U, 62.5 μg)in the hood successively. The NRG solution was drawn into a sterilesyringe. A blunt-tipped needle was exchanged for the syringe and thebubble in the syringe was removed. The pump was held upright and theneedle was inserted through the small opening at the top of the uprightpump until it could go no further. The plunger was pushed slowly to addNRG solution into the pump until the solution began to overflow thepump. The needle was removed and the pump was wiped clean. Thetransparent cap of the flow moderator was taken off to expose a shortstainless steel tube. The steel tube was then inserted into one end of a5 cm PE60 tube. The syringe needle was inserted into another end of thePE60 tube. The plunger of the syringe was pushed to add NRG solution tothe flow moderator until it was full. The long tube of the flowmoderator was then inserted into the pump until its white flangeattached to the pump. The needle was drawn out of the flow moderatorbefore soaking the pump in sterile 0.9% saline at 37° C. overnight.

To install the osmotic pump, Wistar male rats (Shanghai Animal Center ofChinese Academy of Science), each of which weighed 200±20 g, wereanesthetized by intraperitoneally injecting 100 mg/kg (drug/body weight)of ketamine. The area between neck and shoulder of the rats wasdepilated and sanitized. The body was covered with a piece of sterilewet cloth. An incision was then carefully made in the skin between thescapulae to locate and separate the external jugular vein. The distalend of the vein from the heart was ligated. A small hole was made by eyescissors on the wall of the external jugular vein and enlarged bymicroforceps. The PE60 tube connected to the osmotic pump was inserted 2cm into the vein through the hole. The proximal end of the vein from theheart was then bound with PE60 tube to fix the tube. The distal end ofthe vein surrounding the PE60 tube was tied tight to further fix thetube. Using a hemostat, a tunnel was formed by blunt separation of theskin from the incision to scapula. A pocket was finally made on the backof the rat in the midscapular region by spreading the skin further. Thepump was slid through the tunnel into the pocket with the flow moderatorpointing away from the incision. The skin incision was then closed witha suture. The rats were put back into the animal room after revival andwere fed as usual.

After MI rats were treated with NRG through the osmotic pump for 7 days,the rats were sacrificed and their left ventricles were taken and sentto Afflymetrix, Inc. for gene expression analysis. The rat leftventricle was then homogenized and total mRNA was extracted from thehomogenate. The mRNA sample was then studied by Affymetrix Ratexpression array 230 2.0 and the mRNA level of genes were examined usinga microarray. The calculated levels of mRNA corresponding to proteinsrelated to myosin light chain kinase are listed in Table 3. Each datapoint represents the average expression levels from 3 rats.

TABLE 3 Relative Levels Of mRNAs Encoding Proteins Related To MyosinLight Chain Kinase From Rat Left Ventricle Treated with NRG Probe Normalrats MI rats with MI rats with set ID with vehicle vehicle NRG Gene1371541 0.921 ± 0.085 0.951 ± 0.125 1.147 ± 0.165 Myosin, lightpolypeptide kinase (predicted) 1376789 0.997 ± 0.066 0.679 ± 0.098 1.696± 0.189 Similar to Myosin light chain kinase 2, skeletal/cardiac muscle(predicted) 1382239 0.886 ± 0.218 0.591 ± 0.246 1.721 ± 0.339 Similar tomyosin light chain kinase 2, skeletal/cardiac muscle (predicted) 13848180.908 ± 0.296 0.598 ± 0.227 0.335 ± 0.162 Myosin, light polypeptidekinase (predicted) 1386200 0.969 ± 0.274 0.717 ± 0.104 0.946 ± 0.098Similar to Myosin light chain kinase 2, skeletal/cardiac muscle(predicted) 1398820 0.942 ± 0.185 1.115 ± 0.101 0.592 ± 0.195 myosinlight chain kinase 2, skeletal muscle 1398821 0.700 ± 0.254 1.287 ±0.375 0.738 ± 0.217 myosin light chain kinase 2, skeletal muscle

For mRNA sequences hybridizing to probe sets 1376789 and 1382239,expression was increased at least 2-fold in NRG-treated MI rat leftventricle compared to control (vehicle)-treated samples. These resultsdemonstrate that NRG significantly enhances the level of mRNAs that bindwith probe set 1376789 and/or 1382239 in MI rat left ventricle.Accordingly, mRNAs that bind to probe set 1376789 or 1382239 likelyencode proteins which are downstream target(s) of neuregulin.

6.2 Example 2 Cloning of Neukinase cDNA from Rat Left Ventricle RNA

Total RNA was extracted from normal rat left ventricle. RNA, primer(GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT (SEQ ID NO:5)) and AMV reversetranscriptase (Promega) were added to the Promega Reverse TranscriptionSystem (cat. #A3500), and reverse transcription was performed accordingto the manufacturer's protocol. After the reaction, an aliquot of thereaction mixture, reverse primer (GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT(SEQ ID NO:5)) and forward primer (ATGTCAGGAGTTTCAGAGGA (SEQ ID NO:6))(based on predicted similarity to myosin light chain kinase 2) wereadded to a PCR master mix (Sinobio) to amplify target cDNA by PCR.Following PCR, the resulting sample was purified by electrophoresis andligated to pUCm-T plasmid (Promega). The plasmid was then sequenced withthe two primers mentioned above (SEQ ID NOS: 5 and 6). The cDNA sequenceis listed as SEQ ID NO: 1, and the corresponding protein amino acidsequence is listed as SEQ ID NO:2. This protein was named neukinase, andits cDNA sequence was further confirmed by alignment with the sequencesfor probe set 1382239 (SEQ ID NO:9) and probe set 1376789 (SEQ ID NO:10) (see Example 1 above). Alignment of these three sequences to the ratgenome revealed that 325 bp of the 5′ end of SEQ ID NO: 9 overlappedwith the 3′ end of the neukinase gene, and 77 bp of the 5′ end of SEQ IDNO: 10 overlapped with the 3′ of SEQ ID NO:9.

6.3 Example 3 Specific Expression of the Neukinase Gene in Rat Heart

Using neukinase cDNA as template, a subsequence of neukinase (SEQ IDNO:8) was synthesized by PCR using a forward primer(ATGTCAGGAGTTTCAGAGGA (SEQ ID NO:6)) and a reverse primer(CTTGAATTCTCACAGTGACGTATCGATGAT (SEQ ID NO:7)). This fragment (SEQ IDNO:8) was then purified and used as a template to synthesize aradiolabeled neukinase cDNA probe. The neukinase cDNA fragment and[α-³²P]dCTP were added to Promega's Prime-a Gene® labeling system(containing DNA polymerase I large fragment and randomhexadeoxyribonucleotides) to synthesize labeled probes. Reactionproducts were loaded onto Sephacryl® S-400 spin columns (Promega), andcolumns were spun to harvest probes longer than 270 bp. Probes were thenused for Northern Blot analysis of Clontech's Rat MTN™ blot, whichincludes poly A⁺ RNA extracted from various rat organs (heart, brain,spleen, lung, liver, skeletal muscle, kidney and testis). FIG. 1 showsthat neukinase specific probe hybridized only with an mRNA ofapproximately 4.4 kb from heart tissue, suggesting that the neukinasegene is a cardiac specific gene. The blot was also hybridized withβ-actin specific probe (Clontech) as a loading control.

6.4 Example 4 Cloning of Human Neukinase cDNA from Human Left VentricleRNA

Total RNA was extracted from human left ventricle. RNA, Oligo dT primerTTTTTTTTTTTTTTT (SEQ ID NO;26))and AMV reverse transcriptase (Promega)were added to the Promega Reverse Transcription System for reversetranscription. After the reaction, an aliquot of the reaction mixture,forward primer (GACACCACCGCCTGAGTGAGAAC (SEQ ID NO: 11)) and reverseprimer (CCATTGGAGCAGCAGAGTTGAAGA (SEQ ID NO: 12)) was added to a PCRmaster mix (Sinobio), and PCR was performed to amplify target cDNA.After the reaction, the resulting mixtures were purified byelectrophoresis and ligated to pUCm-T plasmid (Promega) and sequenced.The human neukinase cDNA sequence is listed as SEQ ID NO:4. Putativealternative translation start sites were identified at positions 139 and211 of SEQ ID NO:4, translation from which results in polypeptides of795 amino acids (SEQ ID NO:2) and 819 amino acids (SEQ ID NO:25),respectively.

6.5 Example 5 Human Neukinase Antibody Production

Rabbit polyclonal antibodies against a human neukinase-GST fusionprotein were generated. Briefly, human neukinase cDNA, forward primer(CGCGGATCCATGGACACAAAGCTGAACATG (SEQ ID NO: 13)) and reverse primer(CCTTAAGTCACGTGGCCCCCACCAAAGCGAT (SEQ ID NO: 14)) were added to a PCRmaster mix (Sinobio), and PCR was performed. After the PCR, theresulting mixtures were purified by electrophoresis. Both the purifiedDNA and pGEX-2T plasmid (GE healthcare) were digested by BamHI and EcoRIrespectively before ligation. The cDNA sequence of human neukinasefragment is listed as SEQ ID NO: 15, and the amino acid sequence of thefragment is shown as SEQ ID NO: 16.

The ligated construct containing human neukinase fragment cDNA wastransformed into BL21 cells before IPTG was added to the culture toinduce high expression of neukinase fragment. The cells were collectedby centrifugation of the culture before sonication. The sonicated cellsuspension was further centrifugated to pellet inclusion bodies. Afterremoval of the supernatant, 8M urea was added to dissolve the inclusionbodies. The neukinase fragment solution was then dialysed to remove ureaand to simultaneously refold the fragment. The fragment was thenpurified by GST affinity column and hypodermically injected to Rabbit toproduce antibody. After 2 weeks, rabbit serum was drawn for purificationof antibody.

6.6 Example 6 Specific Expression of Neukinase in Human Heart Tissue

Tissues from human gut, liver, heart, skeletal muscle, lung, kidney,uterus, spleen and thyroid were homogenized separately and lysed withlysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mMEDTA, 50 mM NaF, 2 mM Sodium Vanadate, 2 mM PMSF, cocktail proteaseinhibitor (Roche, 1 piece for 25 ml)). Protein samples from lysedtissues were subjected to SDS-PAGE, then transferred to a PVDF membranefor Western blot. The expression of neukinase protein in different humantissues was detected by the antibody produced in example 5. The membranewas probed with a GAPDH-specific antibody as a loading control. As shownin FIG. 2, neukinase was only expressed in human heart. This resultcomplements the mRNA expression of neukinase in rat heart tissue, aspresented in example 3, and further demonstrates that expression ofneukinase is cardiac specific.

6.7 Example 7 Human Neukinase Activity is Calcium and CalmodulinDependent

Expression and Purification of Human Regulatory Myosin Light Chain (RLC)

Total RNA was extracted from human left ventricle tissue. RNA, forwardprimer (GGGAATTCCATATGGCACCTAAGAAAGCAAAGAA (SEQ ID NO: 17)), reverseprimer (CCGCTCGAGGTCCTFTCTTCTCCGTGGGTG (SEQ ID NO: 18)) and AMV reversetranscriptase were added to the Promega Reverse Transcription System forreverse transcription. After the reaction, double stranded cDNA wasligated to pet22b plasmid. The ligated construct was then transformedinto BL21 cells before IPTG was added to induce high expression ofhis-tagged RLC. Cells were pelleted by centrifugation before sonicationto release inclusion bodies. Inclusion bodies were collected by furthercentrifugation before being dissolved by 8M urea. Denatured his-taggedRLC was purified by nickel column and refolded by dialysis to removeurea. The amino acid sequence of RLC corresponds with SEQ ID NO: 19.

Recombinant Expression and Purification of Neukinase

Neukinase cDNA, forward primer(CATCATCTGGTCCGCGTGGATCTATGTCAGGAACCTCCAAGGAGAGT (SEQ ID NO: 20)),reverse primer (CGGAATTCCCATTGGAGCAGCAGAGTTGAAG (SEQ ID NO:21)) and PfuTurbo® DNA polymerase (Stratagene) were added to the PCR reaction systemfor PCR. Following several rounds of amplification, an aliquot of thereaction mixture, new forward primer(CGGGATCCATGCATCATCATCATCATCATCTGGTTCCGCGT (SEQ ID NO:22)), reverseprimer (CGGAATTCCCATTGGAGCAGCAGAGTTGAAGA (SEQ ID NO:21) and PfuTurbo®DNA polymerase (Stratagene) were added to PCR reaction system foradditional rounds of PCR. DNA in the reaction mixture was separated byelectrophoresis, and target DNA encoding neukinase was purified andligated to pcDNA3 plasmid. Ligation reaction products were transformedinto DH5α cells for amplification and sequencing. Clones containing thecorrect construct were amplified in scale-up cultures, and plasmid DNAcontaining neukinase cDNA was extracted using the Qiagen Plasmid MaxiKit. Purified pcDNA3/neukinase plasmid was then transfected into COS7cells using Lipofectamine™ 2000 (Invitrogen). After an initial change ofmedia several hours following transfection, cells were incubated for 48hours at 37° C. Cells were then lysed and harvested using lysis buffer(50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaF,2 mM Sodium Vanadate, 2 mM PMSF, cocktail protease inhibitor (Roche, 1tablet for 25 ml lysis buffer)). The cell suspension was centrifuged at12000 g for 20 minutes, and resuspended pellets were filtered through a0.45 μm membrane (Millipore). The sample was then mixed with His-tagantibody (Beyotime) and 50% Protein A Sepharose 4 Fast Flow in lysisbuffer and incubated on ice for 3 hours with gentle shaking. The mixturewas centrifugated at 12000 g for 20 seconds before the supernatant wasremoved. The pellet was then washed three times with lysis buffer.Following the last wash, the pellet was resuspended in 1 ml of reactionbuffer (20 mM Tris, pH 7.5, 60 mM KCl), mixed and incubated on ice forfive minutes. The mixture was then centrifuged, the supernatant removed,and another 300 μl reaction buffer was added.

Calcium and Calmodulin-Dependent Phosphorylation of RLC by Neukinase

In-vitro phosphorylation assays were performed utilizing purifiedneukinase, RLC, and Calmodulin (Calbiochem) to determine whetherneukinase phosphorylation of RLC is both calcium and calmodulindependent. The activity of neukinase was assessed in vitro, both in thepresence and absence of Ca2+ and calmodulin, by monitoring the amount ofRLC phosphorylation, as determined by Western blotting forphosphorylated RLC (RLC-P). Three experiments were performedsimultaneously. In experiment 1, the reaction components includedneukinase, ATP, RLC, Ca²⁺, CaM. In experiment 2, the reaction componentsincluded neukinase, ATP, RLC. CaM, but not calcium, and EGTA was addedto chelate calcium in the reaction buffer. In experiment 3, the reactioncomponents included neukinase, ATP, RLC, and Ca²⁺, but not calmodulin.The concentration of reactants, when included in the reaction, were asfollows: 2 mM ATP, 2.5 μM RLC, 0.3 μM Ca²⁺, 1 μM CaM, with or without 2mM EGTA. Phosphorylation reactions were carried out at room temperaturefor 2 hours with gentle shaking. A 20 μl aliquot of each reaction wasremoved, subjected to polyacrylamide gel electrophoresis, andtransferred to a PVDF membrane for Western blot analysis. RLC-P antibody(Cell Signaling) was used to detect RLC-P; the results are presented inFIG. 3.

RLC is highly phosphorylated when neukinase is combined with RLC in thepresence of both Ca²⁺ and calmodulin (lane 1). In contrast, RLCphosphorylation is barely detectable in the absence of Ca²⁺ combinedwith the addition of EGTA to the reaction solution (lane 2). Similarly,RLC phosphorylation is undetectable in the absence of calmodulin (lane3). Taken together, these results indicate that neukinasephosphorylation of RLC is highly dependent on the presence of Ca²⁺ andcalmodulin. Thus, it is believed that the neukinase phosphorylation ofRLC occurs in the following manner:

In the formula, CaM stands for Calmodulin. RLC stands for regulatorymyosin light chain, and RLC-P stands for phosphorylated RLC.

6.8 Example 8 The Activity of Human Neukinase Expressed in Insect Cells

Preparation of Bacmid Containing Human Neukinase cDNA

pcDNA3/neukinase plasmid DNA from example 7 was digested with BamHI andEcoRI to excise the neukinase cDNA fragment. Digestion products wereseparated by electrophoresis, and the human neukinase cDNA fragment wasgel purified and subsequently ligated to pFastBac plasmid DNA digestedwith EcoRI BamHI. DH5α competent cells were transformed with theligation products, plated, and incubated overnight. Mini-prep DNAisolated from several overnight colonies were sent to Invitrogen forsequencing to identify neukinase positive clones. Colonies harboringpFastBac/neukinase plasmids containing the correct neukinase cDNAsequence were amplified further, and plasmid DNA was purified using aPlasmid Maxi Kit (Qiagen). pFastBac/neukinase plasmid DNA was thentransformed into DH10Bac cells, and the cells were inoculated onto anagarose plate containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10μg/ml tetracycline, 200 μg/ml X-gal and 40 μg/ml IPTG and incubated at37° C. for 48 hours. A white colony was picked out and inoculated againonto a new agarose plate containing 50 μg/ml kanamycin, 7 μg/mlgentamicin, 10 g/ml tetracycline, 200 μg/ml X-gal and 40 μg/ml IPTG andincubated at 37° C. overnight. A white colony was then inoculated intoliquid media containing 50 μg/ml kanamycin, 7 μg/ml gentamicin and 10μg/ml tetracycline and gently shaken overnight at 37° C. 6 ml of theliquid culture was taken and centrifuged at 14000 g for 1 min. Thesupernatant was removed, and 1.2 ml of solution 1 (15 mM Tris-HCl,pH8.0, 10 mM EDTA, 100 μg/ml RNase A) was added and mixed gently toresuspend the cells. 1.2 ml of solution 2 (0.2N NaOH, 1% SDS) was thenadded and mixed gently at room temperature for 5 minutes. 1.2 ml 3Mpotassium acetate, pH 5.5 was added slowly while shaking, and themixture was centrifugated at 14000 g for 10 minutes. The supernatant wastransferred to a tube containing 3.2 ml isopropanol. The tube wasinverted several times and left on ice for 6 minutes beforecentrifugation at 14000 g for 15 min. The supernatant was removedcarefully without disturbing the pellet. 2 ml of 70% ethanol was addedto the pellet before the tube was turned upside down several times andcentrifuged at 14000 g for 5 minutes. The tube was left open at roomtemperature for 5-10 min and the pellet allowed to following removal ofresidual supernatant. 40 l TE buffer, pH8.0 was added to dissolve thepurified Bacmid DNA.

Purified Bacmid DNA, forward primer (GTTTTCCCAGTCACGAC (SEQ ID NO:23),also M13+) and reverse primer (CGGAATTCCCATTGGAGCAGCAGAGTTGAAGA (SEQ IDNO: 24)) were added to a PCR master mix (Sinobio) for PCR. The reactionmixture was then electrophoresed to detect the positive clone.

Expression and Purification of Human Neukinase

5.4×10⁶ sf9 insect cells were seeded on a 10 cm plate in Grace's insectmedium (Invitrogen) and left at room temperature for 1 hour. During thistime, 24 μg of Bacmid containing human neukinase cDNA (Bacmid/neukinase)was added to 1.5 ml Grace's medium (without antibiotics and FBS) andmixed. 60 μl Lipofectamine™ 2000 (Invitrogen) was mixed with 1.5 mlGrace's medium (without antibiotics and FBS) and incubated at roomtemperature for 5 min. The Bacmid/neukinase-containing solution wasmixed with the diluted Lipofectamine™ 2000 and incubated at roomtemperature for 20 min. 2 ml Grace's medium (without antibiotics andFBS) was added, and the entire solution was added to sf9 cells followingreplacement of the medium. After 5 hours of incubation, the medium wasremoved at 27° C. and replaced with 10 ml Grace's medium (with 100 Ustreptomycin, 100 U ampicillin and 10% FBS). Medium was collected after72 hours incubation at 27° C. The medium was centrifuged for 5 minutesat 500 g, and the supernatant containing virus was stored at 4° C. inthe dark for short-time storage and at −80° C. for long-term storage.

Virus-containing solution was added to Sf9 cells that were allowed toattach to tissue culture plastic for 1 hour. After the cells wereincubated in virus-containing medium at 27° C. for 72 hours, the mediumwas collected, and a small amount was added to a 100 ml Sf9 cellsuspension in a bottle (2×10⁶ cells/ml). The suspension was incubated at27° C. for 84 hours with shaking (shaking speed: 130 rpm). Afterincubation, the cell suspension was collected and centrifuged at 1000rpm for 10 minutes, and the supernatant was removed. Lysis buffer (50 mMTris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 2 mMSodium Vanadate, 2 mM PMSF, cocktail protease inhibitor (Roche, 1 piecefor 25 ml)) was then added to the cell pellet and the cell suspensionwas sonicated before centrifugation at 12000 rpm for 20 minutes. Thesupernatant was then filtered before loading onto a Nickel column (Nisepharose high performance, GE) to purify human neukinase. The proteinsolution was loaded onto a gel filtration column (HiTrap Desaltingcolumn. GE) to further purify the protein, and the protein was washedfrom the column with buffer containing 50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 0.5 mM EDTA, 0.02%0/Triton X-100, 2 mM DTT, 20% glycerol. Thehuman neukinase solution was aliquoted and stored at −80° C.

Assessment of Human Neukinase Activity

The following provides an exemplary method for determining neukinaseactivity in-vitro. Phosphorylation of RLC by neukinase involves thefollowing reactions and reaction products:

In the formula, PEP stands for phosphoenolpyruvate; PK stands forpyruvate kinase; β-NADH stands for β-Nicotinamide Adenine Dinucleotide(reduced form); LDH stands for Lactic Dehydrogenase; and β-NAD standsfor β-Nicotinamide Adenine Dinucleotide (oxidized form).

Thus, neukinase activity can be determined by measuring the rate ofdecrease of NADH absorbance at 340 nm, which is proportional to the rateof steady-state ATP hydrolysis by neukinase. This assay can also be usedto detect agents which can enhance or inhibit the activity of neukinase.In the assay, an 800 μl reaction comprises: 20 mM Tris, pH 7.5, 60 mMKCl, 1 mM DTT, 3.75 mM MgCl₂, 1 mM ATP, 0.3 μM CaCl2, 1.5 mM PEP, 20U/ml PK, 20 U/ml LDH, 90 μM RLC, 250 μM β-NADH, 1 μM CaM and 100 nMneukinase, ΔOD/min/nmol nukinase=0.0152/min/nmol neukinase.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of modulating neukinase gene expressionin a cell of a mammal, the method comprising contacting the cell with anisolated polynucleotide comprising SEQ ID NO:4 or its complementarystrand.
 2. The method of claim 1, wherein the polynucleotide encodes apolypeptide having SEQ ID NO:2 or SEQ ID NO:25.
 3. The method of claim1, wherein the neukinase gene expression is increased.
 4. The method ofclaim 3, wherein the increased neukinase gene expression increases thelevel of expression of a polypeptide of SEQ ID NO:2 or SEQ ID NO:25. 5.The method of claim 1, wherein the isolated polynucleotide consists ofSEQ ID NO:4 or its complementary strand.
 6. The method of claim 1,wherein the cell is a cardiac cell.
 7. The method of claim 1, whereinthe modulation is in vitro modulation.